Provided by: vsearch_2.22.1-1_amd64 
NAME
vsearch — a versatile open-source tool for microbiome analysis, including chimera
detection, clustering, dereplication and rereplication, extraction, FASTA/FASTQ/SFF file
processing, masking, orienting, pairwise alignment, restriction site cutting, searching,
shuffling, sorting, subsampling, and taxonomic classification of amplicon sequences for
metagenomics, genomics, and population genetics.
SYNOPSIS
Chimera detection:
vsearch (--uchime_denovo | --uchime2_denovo | --uchime3_denovo) fastafile
(--chimeras | --nonchimeras | --uchimealns | --uchimeout) outputfile [options]
vsearch --uchime_ref fastafile (--chimeras | --nonchimeras | --uchimealns |
--uchimeout) outputfile --db fastafile [options]
Clustering:
vsearch (--cluster_fast | --cluster_size | --cluster_smallmem | --cluster_unoise)
fastafile (--alnout | --biomout | --blast6out | --centroids | --clusters |
--mothur_shared_out | --msaout | --otutabout | --profile | --samout | --uc |
--userout) outputfile --id real [options]
Dereplication and rereplication:
vsearch --fastx_uniques (fastafile | fastqfile) (--fastaout | --fastqout |
--tabbedout | --uc) outputfile [options]
vsearch (--derep_fulllength | --derep_id | --derep_prefix) fastafile (--output |
--uc) outputfile [options]
vsearch --derep_smallmem (fastafile | fastqfile) --fastaout outputfile [options]
vsearch --rereplicate fastafile --output outputfile [options]
Extraction of sequences:
vsearch --fastx_getseq fastafile (--fastaout | --fastqout | --notmatched |
--notmatchedfq) outputfile --label label [options]
vsearch --fastx_getseqs fastafile (--fastaout | --fastqout | --notmatched |
--notmatchedfq) outputfile (--label label --labels labelfile | --label_word label
| --label_words labelfile) [options]
vsearch --fastx_getsubseq fastafile (--fastaout | --fastqout | --notmatched |
--notmatchedfq) outputfile --label label [--subseq_start position] [--subseq_end
position] [options]
FASTA/FASTQ/SFF file processing:
vsearch --fasta2fastq fastqfile --fastqout outputfile [options]
vsearch --fastq_chars fastqfile [options]
vsearch --fastq_convert fastqfile --fastqout outputfile [options]
vsearch (--fastq_eestats | --fastq_eestats2) fastqfile --output outputfile
[options]
vsearch --fastq_filter fastqfile [--reverse fastqfile] (--fastaout |
--fastaout_discarded | --fastqout | --fastqout_discarded --fastaout_rev |
--fastaout_discarded_rev | --fastqout_rev | --fastqout_discarded_rev) outputfile
[options]
vsearch --fastq_join fastqfile --reverse fastqfile (--fastaout | --fastqout)
outputfile [options]
vsearch --fastq_mergepairs fastqfile --reverse fastqfile (--fastaout | --fastqout |
--fastaout_notmerged_fwd | --fastaout_notmerged_rev | --fastqout_notmerged_fwd |
--fastqout_notmerged_rev | --eetabbedout) outputfile [options]
vsearch --fastq_stats fastqfile [--log logfile] [options]
vsearch --fastx_filter inputfile [--reverse inputfile] (--fastaout |
--fastaout_discarded | --fastqout | --fastqout_discarded --fastaout_rev |
--fastaout_discarded_rev | --fastqout_rev | --fastqout_discarded_rev) outputfile
[options]
vsearch --fastx_revcomp inputfile (--fastaout | --fastqout) outputfile [options]
vsearch --sff_convert sff-file --fastqout outputfile [options]
Masking:
vsearch --fastx_mask fastxfile (--fastaout | --fastqout) outputfile [options]
vsearch --maskfasta fastafile --output outputfile [options]
Orienting:
vsearch --orient fastxfile --db fastafile (--fastaout | --fastqout | --notmatched |
--tabbedout) outputfile [options]
Pairwise alignment:
vsearch --allpairs_global fastafile (--alnout | --blast6out | --matched |
--notmatched | --samout | --uc | --userout) outputfile (--acceptall | --id real)
[options]
Restriction site cutting:
vsearch --cut fastafile --cut_pattern pattern (--fastaout | --fastaout_rev |
--fastaout_discarded | --fastaout_discarded_rev) outputfile [options]
Searching:
vsearch --search_exact fastafile --db fastafile (--alnout | --biomout | --blast6out
| --mothur_shared_out | --otutabout | --samout | --uc | --userout | --lcaout)
outputfile [options]
vsearch --usearch_global fastafile --db fastafile (--alnout | --biomout |
--blast6out | --mothur_shared_out | --otutabout | --samout | --uc | --userout |
--lcaout) outputfile --id real [options]
Shuffling and sorting:
vsearch (--shuffle | --sortbylength | --sortbysize) fastafile --output outputfile
[options]
Subsampling:
vsearch --fastx_subsample fastafile (--fastaout | --fastqout) outputfile
(--sample_pct real | --sample_size positive integer) [options]
Taxonomic classification:
vsearch --sintax fastafile --db fastafile --tabbedout outputfile [--sintax_cutoff
real] [options]
UDB database handling:
vsearch --makeudb_usearch fastafile --output outputfile [options]
vsearch --udb2fasta udbfile --output outputfile [options]
vsearch (--udbinfo | --udbstats) udbfile [options]
DESCRIPTION
Environmental or clinical molecular diversity studies generate large volumes of amplicons
(e.g.; SSU-rRNA sequences) that need to be checked for chimeras, dereplicated, masked,
sorted, searched, clustered or compared to reference sequences. The aim of vsearch is to
offer a all-in-one open source tool to perform these tasks, using optimized algorithm
implementations and harvesting the full potential of modern computers, thus providing fast
and accurate data processing.
Comparing nucleotide sequences is at the core of vsearch. To speed up comparisons, vsearch
implements an extremely fast Needleman-Wunsch algorithm, making use of the Streaming SIMD
Extensions (SSE2) of post-2003 x86-64 CPUs. If SSE2 instructions are not available,
vsearch exits with an error message. On Power8 CPUs it will use AltiVec/VSX/VMX
instructions, and on ARMv8 CPUs it will use Neon instructions. Memory usage increases
rapidly with sequence length: for example comparing two sequences of length 1 kb requires
8 MB of memory per thread, and comparing two 10 kb sequences requires 800 MB of memory per
thread. For comparisons involving sequences with a length product greater than 25 million
(for example two sequences of length 5 kb), vsearch uses a slower alignment method
described by Hirschberg (1975) and Myers and Miller (1988), with much smaller memory
requirements.
Input
vsearch accept as input fasta or fastq files containing one or several nucleotidic
entries. In fasta files, each entry is made of a header and a sequence. The header is
defined as the string comprised between the initial '>' symbol and the first space, tab or
the end of the line, unless the --notrunclabels option is in effect, in which case the
entire line is included. The header should contain printable ascii characters (33-126).
The program will terminate with a fatal error if there are unprintable ascii characters. A
warning will be issued if non-ascii characters (128-255) are encountered.
If the header matches '>[;]size=integer;label', vsearch interpret integer as the number of
occurrences (or abundance) of the sequence in the study. That abundance information is
used or created during chimera detection, clustering, dereplication, sorting and
searching.
The sequence is defined as a string of IUPAC symbols (ACGTURYSWKMDBHVN), starting after
the end of the identifier line and ending before the next identifier line, or the file
end. vsearch silently ignores ascii characters 9 to 13, and exits with an error message if
ascii characters 0 to 8, 14 to 31, '.' or '-' are present. All other ascii or non-ascii
characters are stripped and complained about in a warning message.
In fastq files, each entry is made of sequence header starting with a symbol '@', a
nucleotidic sequence (same rules as for fasta sequences), a quality header starting with a
symbol '+' and a string of ASCII characters (offset 33 or 64), each one encoding the
quality value of the corresponding position in the nucleotidic sequence.
vsearch operations are case insensitive, except when soft masking is activated. Masking is
automatically applied during chimera detection, clustering, masking, pairwise alignment
and searching. Soft masking is specified with the options '--dbmask soft' (for searching
and chimera detection with a reference) or '--qmask soft' (for searching, de novo chimera
detection, clustering and masking). When using soft masking, lower case letters indicate
masked symbols, while upper case letters indicate regular symbols. Masked symbols are
never included in the unique index words used for sequence comparisons, otherwise they are
treated as normal symbols.
When comparing sequences during chimera detection, dereplication, searching and
clustering, T and U are considered identical, regardless of their case. When aligning
sequences, identical symbols will receive a positive match score (default +2). If two
symbols are not identical, their alignment result in a negative mismatch score (default
-4). Aligning a pair of symbols where at least one of them is an ambiguous symbol
(BDHKMNRSVWY) will always result in a score of zero. Alignment of two identical ambiguous
symbols (for example, R vs R) also receives a score of zero. When computing the amount of
similarity by counting matches and mismatches after alignment, ambiguous nucleotide
symbols will count as matching to other symbols if they have at least one of the
nucleotides (ACGTU) they may represent in common. For example: W will match A and T, but
also any of MRVHDN. When showing alignments (for example with the --alnout option) matches
involving ambiguous symbols will be shown with a plus character (+) between them while
exact matches between non-ambiguous symbols will be shown with a vertical bar character
(|).
vsearch can read data from standard files and write to standard files, but it can also
read from pipes and write to pipes! For example, multiple fasta files can be piped into
vsearch for dereplication. To do so, file names can be replaced with:
- the symbol '-', representing '/dev/stdin' for input files or '/dev/stdout' for
output files,
- a named pipe created with the command mkfifo,
- a process substitution '<(command)' as input or '>(command)' as output.
vsearch can automatically read compressed gzip or bzip2 files if the appropriate libraries
are present during the compilation. vsearch can also read pipes streaming compressed gzip
or bzip2 data if the options --gzip_decompress or --bzip2_decompress are selected. When
reading from a pipe, the progress indicator is not updated.
Options
vsearch recognizes a large number of command-line commands and options. For easier
navigation, options are grouped below by theme (chimera detection, clustering,
dereplication and rereplication, FASTA/FASTQ file processing, masking, pairwise alignment,
searching, shuffling, sorting, and subsampling). We start with the general options that
apply to all themes. Options start with a double dash (--). A single dash (-) may also be
used, except on NetBSD systems. Option names may be shortened as long as they are not
ambiguous (e.g. --derep_f).
Help and version commands:
--help --h
Display help text with brief information about all commands and options.
--version --v
Output version information and a citation for the VSEARCH publication.
Show the status of the support for gzip- and bzip2-compressed input files.
General options:
--bzip2_decompress
When reading from a pipe streaming bzip2-compressed data, decompress the
data. This option is not needed when reading from a standard
bzip2-compressed file.
--fasta_width positive integer
Fasta files produced by vsearch are wrapped (sequences are written on
lines of integer nucleotides, 80 by default). Set the value to zero to
eliminate the wrapping.
--gzip_decompress
When reading from a pipe streaming gzip-compressed data, decompress the
data. This option is not needed when reading from a standard gzip-
compressed file.
--label_suffix string
When writing FASTA or FASTQ files, add the suffix string to sequence
headers.
--log filename
Write messages to the specified log file. Information written includes
program version, amount of memory available, number of cores and command
line options, and if need be, informational messages, warnings and fatal
errors. The start and finish times are also recorded as well as the
elapsed time and the maximum amount of memory consumed. The different
vsearch commands can also write additional information to the log file.
--maxseqlength positive integer
All vsearch operations discard sequences longer than integer (50,000
nucleotides by default).
--minseqlength positive integer
All vsearch operations discard sequences shorter than integer: 1
nucleotide by default for sorting or shuffling, 32 nucleotides for
clustering and dereplication as well as the commands --makeudb_usearch,
--sintax, and --usearch_global.
--no_progress
Do not show the gradually increasing progress indicator.
--notrunclabels
Do not truncate sequence labels at first space or tab, but use the full
header in output files. Turned off by default for all commands except the
sintax command.
--quiet Suppress all messages to stdout and stderr except for warnings and fatal
error messages.
--sample string
When writing FASTA or FASTQ files, add the the given sample identifier
string to sequence headers. For instance, if the given string is ABC, the
text ";sample=ABC" will be added to the header.
--threads positive integer
Number of computation threads to use (1 to 1024). The number of threads
should be less than or equal to the number of available CPU cores. The
default is to use all available resources and to launch one thread per
core. The following commands are multi-threaded: allpairs_global,
cluster_fast, cluster_size, cluster_smallmem, cluster_unoise,
fastq_mergepairs, fastx_mask, maskfasta, search_exact, sintax, uchime_ref,
and usearch_global. Only one thread is used for the other commands.
Chimera detection options:
Chimera detection is based on a scoring function controlled by five options (--dn,
--mindiffs, --mindiv, --minh, --xn). Sequences are first sorted by decreasing
abundance, if available, and compared on their plus strand only (case insensitive).
Input sequences are masked as specified with the --qmask and --hardmask options.
Masking of the database for reference based chimera detection is specified with the
--dbmask option.
In de novo mode, input fasta file must present abundance annotations (i.e. a
pattern [;]size=integer[;] in the fasta header). Input order matters for chimera
detection, so we recommend to sort sequences by decreasing abundance (default of
--derep_fulllength command). If your sequence set needs to be sorted, please see
the --sortbysize command in the sorting section.
--abskew real
When using --uchime_denovo, the abundance skew is used to distinguish in a
three-way alignment which sequence is the chimera and which are the
parents. The assumption is that chimeras appear later in the PCR
amplification process and are therefore less abundant than their parents.
For --uchime3_denovo the default value is 16.0. For the other commands,
the default value is 2.0, which means that the parents should be at least
2 times more abundant than their chimera. Any positive value equal or
greater than 1.0 can be used.
--alignwidth positive integer
When using --uchimealns, set the width of the three-way alignments (80
nucleotides by default). Set to zero to eliminate wrapping.
--borderline filename
Output borderline chimeric sequences to filename, in fasta format.
Borderline chimeric sequences are sequences that have a high enough score
but which are not sufficiently different from their closest parent.
--chimeras filename
Output chimeric sequences to filename, in fasta format. Output order may
vary when using multiple threads.
--db filename
When using --uchime_ref, detect chimeras using the reference sequences
contained in filename. Reference sequences are assumed to be chimera-free.
Chimeras cannot be detected if their parents, or sufficiently close
relatives, are not present in the database. The file name must refer to a
FASTA file or to a UDB file. If a UDB file is used, it should be created
using the --makeudb_usearch command with the --dbmask dust option.
--dn strictly positive real number
pseudo-count prior on the number of no votes, corresponding to the
parameter n in the chimera scoring function (default value is 1.4).
Increasing --dn reduces the likelihood of tagging a sequence as a chimera
(less false positives, but also more false negatives).
--fasta_score
Add the chimera score to the headers in the fasta output files for
chimeras, non-chimeras and borderline sequences, using the format
';uchime_denovo=float;'.
--mindiffs positive integer
Minimum number of differences per segment (default value is 3). The
parameter is ignored with --uchime2_denovo and --uchime3_denovo.
--mindiv real
Minimum divergence from closest parent (default value is 0.8). The
parameter is ignored with --uchime2_denovo and --uchime3_denovo.
--minh real
Minimum score (h). Increasing this value tends to reduce the number of
false positives and to decrease sensitivity. Default value is 0.28, and
values ranging from 0.0 to 1.0 included are accepted. The parameter is
ignored with --uchime2_denovo and --uchime3_denovo.
--nonchimeras filename
Output non-chimeric sequences to filename, in fasta format. Output order
may vary when using multiple threads.
--relabel string
Relabel sequences using the prefix string and a ticker (1, 2, 3, etc.) to
construct the new headers. Use --sizeout to conserve the abundance
annotations.
--relabel_keep
When relabelling, keep the old identifier in the header after a space.
--relabel_md5
Relabel sequences using the MD5 message digest algorithm applied to each
sequence. Former sequence headers are discarded. The sequence is converted
to upper case and each 'U' is replaced by a 'T' before computation of the
digest. The MD5 digest is a cryptographic hash function designed to
minimize the probability that two different inputs give the same output,
even for very similar, but non-identical inputs. Still, there is a very
small, but non-zero, probability that two different inputs give the same
digest (i.e. a collision). MD5 generates a 128-bit (16-byte) digest that
is represented by 16 hexadecimal numbers (using 32 symbols among
0123456789abcdef). Use --sizeout to conserve the abundance annotations.
--relabel_self
Relabel sequences using each sequence itself as a label.
--relabel_sha1
Relabel sequences using the SHA1 message digest algorithm applied to each
sequence. It is similar to the --relabel_md5 option but uses the SHA1
algorithm instead of the MD5 algorithm. SHA1 generates a 160-bit (20-byte)
digest that is represented by 20 hexadecimal numbers (40 symbols). The
probability of a collision (two non-identical sequences resulting in the
same digest) is smaller for the SHA1 algorithm than it is for the MD5
algorithm.
--self When using --uchime_ref, ignore a reference sequence when its label
matches the label of the query sequence (useful to estimate false-positive
rate in reference sequences).
--selfid When using --uchime_ref, ignore a reference sequence when its nucleotide
sequence is strictly identical to the nucleotidic sequence of the query.
--sizein In de novo mode, abundance annotations (pattern '[>;]size=integer[;]')
present in sequence headers are taken into account by default (--sizein is
always implied). This option is ignored by --uchime_ref.
--sizeout
When relabelling, add abundance annotations to fasta headers (using the
format ';size=integer;').
--uchime_denovo filename
Detect chimeras present in the fasta-formatted filename, without external
references (i.e. de novo). Automatically sort the sequences in filename by
decreasing abundance beforehand (see the sorting section for details).
Multithreading is not supported.
--uchime2_denovo filename
Detect chimeras present in the fasta-formatted filename, using the UCHIME2
algorithm. This algorithm is designed for denoised amplicons (see
--cluster_unoise). Automatically sort the sequences in filename by
decreasing abundance beforehand (see the sorting section for details).
Multithreading is not supported.
--uchime3_denovo filename
Detect chimeras present in the fasta-formatted filename, using the UCHIME2
algorithm. The only difference from --uchime2_denovo is that the default
minimum abundance skew (--abskew) is set to 16.0 rather than 2.0.
--uchime_ref filename
Detect chimeras present in the fasta-formatted filename by comparing them
with reference sequences (option --db). Multithreading is supported.
--uchimealns filename
Write the three-way global alignments (parentA, parentB, chimera) to
filename using a human-readable format. Use --alignwidth to modify
alignment length. Output order may vary when using multiple threads. All
sequences are converted to upper case before alignment. Lower case letters
indicate disagreement in the alignment.
--uchimeout filename
Write chimera detection results to filename using a 18-field,
tab-separated uchime-like format. Use --uchimeout5 to use a format
compatible with usearch v5 and earlier versions. Rows output order may
vary when using multiple threads.
1. score: higher score means a more likely chimeric alignment.
2. Q: query sequence label.
3. A: parent A sequence label.
4. B: parent B sequence label.
5. T: top parent sequence label (i.e. parent most similar to the
query). That field is removed when using --uchimeout5.
6. idQM: percentage of similarity of query (Q) and model (M)
constructed as a part of parent A and a part of parent B.
7. idQA: percentage of similarity of query (Q) and parent A.
8. idQB: percentage of similarity of query (Q) and parent B.
9. idAB: percentage of similarity of parent A and parent B.
10. idQT: percentage of similarity of query (Q) and top parent (T).
11. LY: yes votes in the left part of the model.
12. LN: no votes in the left part of the model.
13. LA: abstain votes in the left part of the model.
14. RY: yes votes in the right part of the model.
15. RN: no votes in the right part of the model.
16. RA: abstain votes in the right part of the model.
17. div: divergence, defined as (idQM - idQT).
18. YN: query is chimeric (Y), or not (N), or is a borderline case
(?).
--uchimeout5
When using --uchimeout, write chimera detection results using a 17-field,
tab-separated uchime-like format (drop the 5th field of --uchimeout),
compatible with usearch version 5 and earlier versions.
--xn strictly positive real number
weight of no votes, corresponding to the parameter beta in the scoring
function (default value is 8.0). Increasing --xn reduces the likelihood of
tagging a sequence as a chimera (less false positives, but also more false
negatives).
--xsize Strip abundance information from the headers when writing the output file.
Clustering options:
vsearch implements a single-pass, greedy centroid-based clustering algorithm,
similar to the algorithms implemented in usearch, DNAclust and sumaclust for
example. Important parameters are the global clustering threshold (--id) and the
pairwise identity definition (--iddef).
Input sequences are masked as specified with the --qmask and --hardmask options.
--biomout filename
Generate an OTU table in the biom version 1.0 JSON file format as
specified at <http://biom-
format.org/documentation/format_versions/biom-1.0.html>. The format
describes how to store a sparse matrix containing the abundances of the
OTUs in the different samples. This format is much more efficient than the
classic and mothur OTU table formats available with the --otutabout and
--mothur_shared_out options, respectively, and is recommended at least for
large tables. The OTUs are represented by the cluster centroids. Taxonomy
information will be included for the OTUs if available. Sample identifiers
will be extracted from the headers of all sequences in the input file. If
the header contains ';sample=abc123;' or ';barcodelabel=abc123;' or a
similar string somewhere, then the given sample identifier (here 'abc123')
will be used. The semicolon is not mandatory at the beginning or end of
the header. The sample identifier may contain any printable character
except semicolons. If no such sample label is found, the identifier in the
initial part of the header will be used, but only letters, digits and
underscores are allowed. OTU identifiers will be extracted from the
headers of the cluster centroid sequences. If the header contains
';otu=def789;' or a similar string somewhere, then the given OTU
identifier (here 'def789') will be used. The semicolon is not mandatory at
the beginning or end of the header. The OTU identifier may contain any
printable character except semicolons. If no such OTU label is found, the
identifier in the initial part of the header will be used, and all
characters except semicolons are allowed. Alternatively, OTU identifiers
can be generated using the relabelling options (--relabel, --relabel_self,
--relabel_sha1, or --relabel_md5). Taxonomy information, if present, will
also be extracted from the headers of the centroid sequences. If the
header contains ';tax=Homo_sapiens;' or a similar string somewhere, then
the given taxonomy information (here 'Homo_sapiens') will be used. The
semicolon is not mandatory at the beginning or end of the header. The
taxonomy information may contain any printable character except
semicolons. If an OTU table in the biom version 2.1 HDF5 file format is
required, the biom utility may be used as described at <http://biom-
format.org/documentation/biom_conversion.html>.
--centroids filename
Output cluster centroid sequences to filename, in fasta format. The
centroid is the sequence that seeded the cluster (i.e. the first sequence
of the cluster).
--clusterout_id
Add cluster identifier information to the output files when using the
--centroids, --consout and --profile options.
--clusterout_sort
Sort some output files by decreasing abundance instead of input order. It
applies to the --consout, --msaout, --profile, --centroids, and --uc
options. For --uc, the sorting applies only to the centroid information
part (the C lines).
--cluster_fast filename
Clusterize the fasta sequences in filename, automatically sort by
decreasing sequence length beforehand.
--cluster_size filename
Clusterize the fasta sequences in filename, automatically sort by
decreasing sequence abundance beforehand.
--cluster_smallmem filename
Clusterize the fasta sequences in filename without automatically modifying
their order beforehand. Sequence are expected to be sorted by decreasing
sequence length, unless --usersort is used.
--cluster_unoise filename
Perform denoising of the fasta sequences in filename according to the
UNOISE version 3 algorithm by Robert Edgar, but without the chimera
removal step. The options --minsize (default 8) and --unoise_alpha
(default 2.0) may be specified. Chimera removal (de novo) should be
performed afterwards with --uchime3_denovo.
--clusters string
Output each cluster to a separate fasta file using the prefix string and a
ticker (0, 1, 2, etc.) to construct the path and filenames.
--consout filename
Output cluster consensus sequences to filename. For each cluster, a
multiple alignment is computed, and a consensus sequence is constructed by
taking the majority symbol (nucleotide or gap) from each column of the
alignment. Columns containing a majority of gaps are skipped, except for
terminal gaps. If the --sizein option is specified, sequence abundances
will be taken into account.
--cons_truncate
This command is ignored. A warning is issued.
--id real
Do not add the target to the cluster if the pairwise identity with the
centroid is lower than real (value ranging from 0.0 to 1.0 included). The
pairwise identity is defined as the number of (matching columns) /
(alignment length - terminal gaps). That definition can be modified by
--iddef.
--iddef 0|1|2|3|4
Change the pairwise identity definition used in --id. Values accepted are:
0. CD-HIT definition: (matching columns) / (shortest sequence
length).
1. edit distance: (matching columns) / (alignment length).
2. edit distance excluding terminal gaps (same as --id).
3. Marine Biological Lab definition counting each gap opening
(internal or terminal) as a single mismatch, whether or not the
gap was extended: 1.0 - [(mismatches + gap openings)/(longest
sequence length)]
4. BLAST definition, equivalent to --iddef 1 in a context of
global pairwise alignment.
--minsize positive integer
Specify the minimum abundance of sequences for denoising using
--cluster_unoise. The default is 8.
--msaout filename
Output a multiple sequence alignment and a consensus sequence for each
cluster to filename, in fasta format. Be warned that vsearch computes
center star multiple sequence alignments using a fast method whose
accuracy can decrease significantly when using low pairwise identity
thresholds. The consensus sequence is constructed by taking the majority
symbol (nucleotide or gap) from each column of the alignment. Columns
containing a majority of gaps are skipped, except for terminal gaps. If
the --sizein option is specified, sequence abundances will be taken into
account when computing the consensus.
--mothur_shared_out filename
Output an OTU table in the mothur 'shared' tab-separated plain text format
as described at <https://www.mothur.org/wiki/Shared_file>. The format
describes how a matrix containing the abundances of the OTUs in the
different samples is stored. The first line will start with the strings
'label', 'group' and 'numOtus' and is followed by a list of all OTU
identifiers. The following lines, one for each sample, starts with the
string 'vsearch' followed by the sample identifier, the total number of
OTUs, and a list of abundances for each OTU in that sample, in the order
given on the first line. The OTU and sample identifiers are extracted from
the FASTA headers of the sequences. The OTUs are represented by the
cluster centroids. See the --biomout option for further details.
--otutabout filename
Output an OTU table in the classic tab-separated plain text format as a
matrix containing the abundances of the OTUs in the different samples. The
first line will start with the string '#OTU ID' and is followed by a tab-
separated list of all sample identifiers. The following lines, one for
each OTU, starts with the OTU identifier and is followed by a tab-
separated list of abundances for that OTU in each sample, in the order
given on the first line. The OTU and sample identifiers are extracted from
the FASTA headers of the sequences. The OTUs are represented by the
cluster centroids. An extra column is added to the right of the table if
taxonomy information is available for at least one of the OTUs. This
column will be labelled 'taxonomy' and each row will then contain the
taxonomy information extracted for that OTU. See the --biomout option for
further details.
--profile filename
Output a sequence profile to a text file with the frequency of each
nucleotide in each position in the multiple alignment for each cluster.
There is a FASTA-like header line for each cluster, followed by the
profile information in a tab-separated format. The eight columns are:
position (0-based), consensus nucleotide, number of As, number of Cs,
number of Gs, number of Ts or Us, number of gap symbols, and finally the
total number of ambiguous nucleotide symbols (B, D, H, K, M, N, R, S, Y, V
or W). All numbers are integers. If the --sizein option is specified,
sequence abundances will be taken into account.
--qmask none|dust|soft
Mask regions in sequences using the dust or the soft methods, or do not
mask (none). Warning, when using soft masking, clustering becomes case
sensitive. The default is to mask using dust.
--qsegout filename
Write the aligned part of each query sequence to filename in FASTA format.
--relabel string
Relabel sequence identifiers in the output files produced by --consout,
--profile and --centroids options. Please see the description of the same
option under Chimera detection for details.
--relabel_keep
When relabelling, keep the old identifier in the header after a space.
--relabel_md5
Relabel sequence identifiers in the output files produced by --consout,
--profile and --centroids options. Please see the description of the same
option under Chimera detection for details.
--relabel_self
Relabel sequence identifiers in the output files produced by --consout,
--profile and --centroids options. Please see the description of the same
option under Chimera detection for details.
--relabel_sha1
Relabel sequence identifiers in the output files produced by --consout,
--profile and --centroids options. Please see the description of the same
option under Chimera detection for details.
--sizein Take into account the abundance annotations present in the input fasta
file (search for the pattern '[>;]size=integer[;]' in sequence headers).
--sizeorder
When an amplicon is close to 2 or more centroids, both within the distance
specified with the --id option, resolve the ambiguity by clustering it
with the centroid having the highest abundance, not necessarily the
closest one. The option only has effect when the value specified with
--maxaccepts is higher than one. The --sizeorder option turns on what is
sometimes referred to as abundance-based greedy clustering (AGC), in
contrast to the default distance-based greedy clustering (DGC).
--sizeout
Add abundance annotations to the output fasta files (add the pattern
';size=integer;' to sequence headers). If --sizein is specified, abundance
annotations are reported to output files, and each cluster centroid
receives a new abundance value corresponding to the total abundance of the
amplicons included in the cluster (--centroids option). If --sizein is not
specified, input abundances are set to 1 for amplicons, and to the number
of amplicons per cluster for centroids.
--strand plus|both
When comparing sequences with the cluster seed, check the plus strand only
(default) or check both strands.
--tsegout filename
Write the aligned part of each target sequence to filename in FASTA
format.
--uc filename
Output clustering results in filename using a tab-separated uclust-like
format with 10 columns and 3 different type of entries (S, H or C). Each
fasta sequence in the input file can be either a cluster centroid (S) or a
hit (H) assigned to a cluster. Cluster records (C) summarize information
(size, centroid label) for each cluster. In the context of clustering, the
option --uc_allhits has no effect on the --uc output. Column content
varies with the type of entry (S, H or C):
1. Record type: S, H, or C.
2. Cluster number (zero-based).
3. Centroid length (S), query length (H), or cluster size (C).
4. Percentage of similarity with the centroid sequence (H), or set
to '*' (S, C).
5. Match orientation + or - (H), or set to '*' (S, C).
6. Not used, always set to '*' (S, C) or to zero (H).
7. Not used, always set to '*' (S, C) or to zero (H).
8. set to '*' (S, C) or, for H, compact representation of the
pairwise alignment using the CIGAR format (Compact
Idiosyncratic Gapped Alignment Report): M (match/mismatch), D
(deletion) and I (insertion). The equal sign '=' indicates that
the query is identical to the centroid sequence.
9. Label of the query sequence (H), or of the centroid sequence
(S, C).
10. Label of the centroid sequence (H), or set to '*' (S, C).
--unoise_alpha real
Specify the alpha parameter to the --cluster_unoise command. The default
is 2.0.
--usersort
When using --cluster_smallmem, allow any sequence input order, not just a
decreasing length ordering.
--xsize Strip abundance information from the headers when writing the output file.
... Most searching options as well as score filtering, gap penalties and
masking also apply to clustering (see the Searching section for
definitions): --alnout, --blast6out, --fastapairs, --matched,
--notmatched, --maxaccept, --maxreject, --samout, --userout, --userfields
Dereplication and rereplication options:
VSEARCH can dereplicate sequences with the commands --derep_fulllength, --derep_id,
--derep_smallmem, --derep_prefix and --fastx_uniques. The --derep_fulllength
command is depreciated and is replaced by the new --fastx_uniques command that can
also handle FASTQ files in addition to FASTA files. The --derep_fulllength,
--derep_smallmem, and --fastx_uniques commands requires strictly identical
sequences of the same length, but ignores upper/lower case and treats T and U as
identical symbols. The --derep_id command requires both identical sequences and
identical headers/labels. The --derep_prefix command will group sequences with a
common prefix and does not require them to be equally long. The --derep_smallmem
uses a much smaller amount of memory when dereplicating than the other files, and
may be a bit slower and cannot read the input from a pipe. It takes both FASTA and
FASTQ files as input but only writes FASTA output to the file specified with the
--fastaout option. The --fastx_uniques command can write FASTQ output (specified
with --fastqout) or FASTA output (specified with --fastaout) as well as a special
tab-separated column text format (with --tabbedout). The other commands can write
FASTA output to the file specified with the --output option. All dereplication
commands, except --derep_smallmem, can write output to a special UCLUST-like file
specified with the --uc option. The --rereplicate command can duplicate sequences
in the input file according to the abundance of each input sequence. Other valid
options are --fastq_ascii, --fastq_asciiout, --fastq_qmax, --fastq_qmaxout,
--fastq_qmin, --fastq_qminout, --fastq_qout_max, --maxuniquesize, --minuniquesize,
--relabel, --relabel_keep, --relabel_md5, --relabel_self, --relabel_sha1, --sizein,
--sizeout, --strand, --topn, and --xsize.
--derep_fulllength filename
Merge strictly identical sequences contained in filename. Identical
sequences are defined as having the same length and the same string of
nucleotides (case insensitive, T and U are considered the same). See the
options --sizein and --sizeout to take into account and compute abundance
values. This command does not support multithreading.
--derep_id filename
Merge strictly identical sequences contained in filename, as with the
--derep_fulllength command, but the sequence labels (identifiers) on the
header line need to be identical too.
--derep_smallmem filename
Merge strictly identical sequences contained in filename, as with the
--derep_fulllength command, but using much less memory. The output is
written to a FASTA file specified with the --fastaout option. The output
is written in the order that the sequences first appear in the input, and
not in decending abundance order as with the other dereplication commands.
It can read, but not write FASTQ files. This command cannot read from a
pipe, it must be a proper file, as it is read twice. Dereplication is
performed with a 128 bit hash function and it is not verified that grouped
sequences are identical, however the probability that two different
sequences are grouped in a dataset of 1 000 000 000 unique sequences is
approximately 1e-21. Multithreading and the options --topn, --uc, or
--tabbedout are not supported.
--derep_prefix filename
Merge sequences with identical prefixes contained in filename. A short
sequence identical to an initial segment (prefix) of another sequence is
considered a replicate of the longer sequence. If a sequence is identical
to the prefix of two or more longer sequences, it is clustered with the
shortest of them. If they are equally long, it is clustered with the most
abundant. Remaining ties are solved using sequence headers and sequence
input order. Sequence comparisons are case insensitive, and T and U are
considered identical. This command does not support multithreading.
--fastaout filename
Write the dereplicated sequences to filename, in fasta format and sorted
by decreasing abundance. Identical sequences receive the header of the
first sequence of their group. If --sizeout is used, the number of
occurrences (i.e. abundance) of each sequence is indicated at the end of
their fasta header using the pattern ';size=integer;'. This option is only
valid for --fastx_uniques and --derep_smallmem.
--fastqout filename
Write the dereplicated sequences to filename, in fastq format and sorted
by decreasing abundance. Identical sequences receive the header of the
first sequence of their group. If --sizeout is used, the number of
occurrences (i.e. abundance) of each sequence is indicated at the end of
their fastq header using the pattern ';size=integer;'. This option is only
valid for --fastx_uniques.
--fastq_ascii positive integer
Define the ASCII character number used as the basis for the FASTQ quality
score. The default is 33, which is used by the Sanger / Illumina 1.8+
FASTQ format (phred+33). The value 64 is used by the Solexa, Illumina 1.3+
and Illumina 1.5+ formats (phred+64). Only 33 and 64 are valid arguments.
--fastq_asciiout positive integer
When using --fastq_convert, --sff_convert or --fasta2fastq, define the
ASCII character number used as the basis for the FASTQ quality score when
writing FASTQ output files. The default is 33. Only 33 and 64 are valid
arguments.
--fastq_qmax positive integer
Specify the maximum quality score accepted when reading FASTQ files. The
default is 41, which is usual for recent Sanger/Illumina 1.8+ files.
--fastq_qmaxout positive integer
Specify the maximum quality score used when writing FASTQ files. The
default is 41, which is usual for recent Sanger/Illumina 1.8+ files. Older
formats may use a maximum quality score of 40.
--fastq_qmin positive integer
Specify the minimum quality score accepted for FASTQ files. The default is
0, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may
use scores between -5 and 2.
--fastq_qminout positive integer
Specify the minimum quality score used when writing FASTQ files. The
default is 0, which is usual for Sanger/Illumina 1.8+ files. Older
versions of the format may use scores between -5 and 2.
--fastq_qout_max
For --fastx_uniques, indicate that the new quality scores computed when
dereplicating FASTQ files should be equal to the maximum (best) of the
input quality scores for each position (corresponding to the lowest error
probability). The default is to output a quality score corresponding to
the average of the error probabilities for each position.
--fastx_uniques filename
Merge strictly identical sequences contained in FASTA or FASTQ file
filename. Identical sequences are defined as having the same length and
the same string of nucleotides (case insensitive, T and U are considered
the same). See the options --sizein and --sizeout to take into account and
compute abundance values. This command does not support multithreading. By
default, the quality scores in FASTQ output files will correspond to the
average error probability of the nucleotides in the each position. If the
--fastq_qout_max option is given, the quality score will be the highest
(best) quality score observed in each position.
--maxuniquesize positive integer
Discard sequences with a post-dereplication abundance value greater than
integer.
--minuniquesize positive integer
Discard sequences with a post-dereplication abundance value smaller than
integer.
--output filename
Write the dereplicated sequences to filename, in fasta format and sorted
by decreasing abundance. Identical sequences receive the header of the
first sequence of their group. If --sizeout is used, the number of
occurrences (i.e. abundance) of each sequence is indicated at the end of
their fasta header using the pattern ';size=integer;'. This option is not
allowed for --fastx_uniques or --derep_smallmem.
--relabel string
Please see the description of the same option under Chimera detection for
details.
--relabel_keep
When relabelling, keep the old identifier in the header after a space.
--relabel_md5
Please see the description of the same option under Chimera detection for
details.
--relabel_self
Please see the description of the same option under Chimera detection for
details.
--relabel_sha1
Please see the description of the same option under Chimera detection for
details.
--rereplicate filename
Duplicate each sequence the number of times indicated by the abundance of
each sequence in the specified file (option --sizein is always implied).
The sequence labels are identical for the same sequence, unless --relabel,
--relabel_self, --relabel_sha1 or --relabel_md5 is used to create unique
labels. Output is written to the file specified with the --output option,
in FASTA format. The output file does not contain abundance information
unless --sizeout is specified, in which case an abundance of 1 is used.
--sizein Take into account the abundance annotations present in the input fasta
file (search for the pattern '[>;]size=integer[;]' in sequence headers).
That option is active by default when rereplicating.
--sizeout
Add abundance annotations to the output fasta file (add the pattern
';size=integer;' to sequence headers). If --sizein is specified, each
unique sequence receives a new abundance value corresponding to its total
abundance (sum of the abundances of its occurrences). If --sizein is not
specified, input abundances are set to 1, and each unique sequence
receives a new abundance value corresponding to its number of occurrences
in the input file.
--strand plus|both
When searching for strictly identical sequences, check the plus strand
only (default) or check both strands.
--tabbedout filename
Output clustering info to the specified tab-separated text file with 6
columns and a row for each input sequence. Column 1 contains the original
label/header of the sequence. Column 2 contains the label of the output
sequence which is equal to the label/header of the first sequence in each
cluster, but potentially relabelled. Column 3 contains the cluster number,
starting from 0. Column 4 contains the sequence number within each
cluster, starting at 0. Column 5 contains the number of sequences in the
cluster. Column 6 contains the original label/header of the first sequence
in the cluster before any potential relabelling. This option is only valid
for the --fastx_uniques command.
--topn positive integer
Output only the top integer sequences (i.e. the most abundant).
--uc filename
Output full-length or prefix-dereplication results in filename using a
tab-separated uclust-like format with 10 columns and 3 different type of
entries (S, H or C). Each fasta sequence in the input file can be either a
cluster centroid (S) or a hit (H) assigned to a cluster. Cluster records
(C) summarize information (size, centroid label) for each cluster. In the
context of dereplication, the option --uc_allhits has no effect on the
--uc output. Column content varies with the type of entry (S, H or C):
1. Record type: S, H, or C.
2. Cluster number (zero-based).
3. Sequence length (S, H), or cluster size (C).
4. Percentage of similarity with the centroid sequence (H), or set
to '*' (S, C).
5. Match orientation + or - (H), or set to '*' (S, C).
6. Not used, always set to '*' (S, C) or 0 (H).
7. Not used, always set to '*' (S, C) or 0 (H).
8. Not used, always set to '*'.
9. Label of the query sequence (H), or of the centroid sequence
(S, C).
10. Label of the centroid sequence (H), or set to '*' (S, C).
--xsize
Strip abundance information from the headers when writing the output file.
Extraction options:
Sequences with headers matching certain criteria can be extracted from FASTA and
FASTQ files using the --fastx_getseq, --fastx_getseqs and --fastx_getsubseq
commands.
The --fastx_getseq command requires the header to match a label specified with the
--label option. If the --label_substr_match option is given, the label may be a
substring located anywhere in the header, otherwise the entire header must match
the label. These matches are not case-sensitive. The headers in the input file are
truncated at the first space or tab character unless the --notrunclabels option is
given. The matching sequences will be written to the files specified with the
--fastaout and --fastqout options, in FASTA and FASTQ format, respectively.
Sequences that do not match are written to the files specified with the
--notmatched and --notmatchedfq options, respectively.
The --fastx_getsubseq command is similar to the --fastx_getseq command, but will
extract a subsequence of the matching sequences. The start position is specified
with the --subseq_start option and the end position is specified with the
--subseq_end option. The positions are 1-based, meaning that the first symbol of
the sequence is at position 1. If the start or end position option is not
specified, the default is to start at the first position and end at the last
position in the sequence.
The --fastx_getseqs command is similar to the --fastx_getseq command but allows
more flexibility in specifying the label(s) to be matched. A single label may be
specified using the --label option as described above. Alternatively, a file
containing a list of labels to be matched may be specified with the --labels
option. The file must be a plain text file with one label on each line. The
--label_word and --label_words options may be used to specify either a single word
or a file containing a list of words, respectively, to be matched. Words are
defined as character sequences delimited either by a character that is not alpha-
numeric (A-Z, a-z, or 0-9) or by the beginning or end of the header. Word matching
is case-sensitive. The --label_field option will limit the matching of words to a
certain field in the header.
--fastaout filename
Write the extracted sequences in FASTA format to the file with the given
name.
--fastqout filename
Write the extracted sequences in FASTQ format to the file with the given
name. This option is illegal if the input is in FASTA format.
--fastx_getseq filename
Extract sequences from the given FASTA or FASTQ file. Specify a label to
match using the --label option. Output files are specified with the
--fastaout, --fastqout, --notmatched and --notmatchedfq options.
--fastx_getseqs filename
Extract sequences from the given FASTA or FASTQ file. Specify the label or
labels to match using one of the following options: --label, --labels,
--label_word, or --label_words. Output files are specified with the
--fastaout, --fastqout, --notmatched and --notmatchedfq options.
--fastx_getsubseq filename
Extract a certain part of some of the sequences in the given FASTA or
FASTQ file. Specify labels to match using the --label option. Specify the
subsequence range to be extracted with the --subseq_start and --subseq_end
options. Output files are specified with the --fastaout, --fastqout,
--notmatched and --notmatchedfq options.
--label string
Specify the label to match in the sequence header. Unless the
--label_substr_match option is given, the label must match the entire
header. The comparison is not case-sensitive.
--label_field string
Specify a field name to be used when matching using the --label_word or
--label_words option. The field name is a string like "abc" that must
precede the word to be matched with an equals sign (=) in between. The
field must be delimited by semicolons or the beginning or end of the
header. The following header will match the label 123 in the field abc:
"seq1;abc=123".
--label_substr_match
The labels specified with the --label or the --labels option may match
anywhere in the header if this option is given. Otherwise a label needs to
match the entire header.
--label_word string
Specify a word to match in the sequence header. Words are defined as
strings delimited by either the start or end of the header or by any
symbol that is not a letter (A-Z, a-z) or digit (0-9). The comparison is
case-sensitive.
--label_words filename
Specify a file containing words to be matched against the sequence
headers. The plain text file must contain one word on each line. Words
are defined as strings delimited by either the start or end of the header
or by any symbol that is not a letter (A-Z, a-z) or digit (0-9). The
comparison is case-sensitive.
--labels filename
Specify a file containing labels to be matched against the sequence
headers. The plain text file must contain one label on each line. Unless
the --label_substr_match option is given, a label must match the entire
header. The comparison is not case-sensitive.
--notmatched filename
Write the sequences that were not extracted to the file with the given
name, in FASTA format.
--notmatchedfq filename
Write the sequences that were not extracted to the file with the given
name, in FASTQ format. This option is illegal if the input is in FASTA
format.
--subseq_end positive integer
Specify the end position in the sequences when extracting subsequences
using the --fastx_getsubseq command. Positions are 1-based, so the
sequences start at position 1. The default is to end at the end of the
sequence if this option is not specified.
--subseq_start positive integer
Specify the starting position in the sequences when extracting
subsequences using the --fastx_getsubseq command. Positions are 1-based,
so the sequences start at position 1. The default is to start at the
beginning of the sequence (position 1), if this option is not specified.
FASTA/FASTQ/SFF file processing options:
Analyse, trim, filter, convert, merge, join or reverse complement sequences in
FASTA, FASTQ or SFF files. The --fastq_chars command can be used to analyse FASTQ
files to identify the quality encoding and the range of quality score values used.
To convert between different FASTQ file variants, use the --fastq_convert command.
Statistical analysis of the quality and length of the sequences in a FASTQ file may
be performed with the --fastq_stats, --fastq_eestats, and --fastq_eestats2
commands. Sequences may be trimmed, filtered and converted by the --fastq_filter
or --fastx_filter commands. The --sff_convert command can be used to convert SFF
files to FASTQ, while the --fasta2fastq command will convert a FASTA file to a
FASTQ file with fake quality scores. Paired-end reads can be merged using the
--fastq_mergepairs command or joined with the --fastq_join command. The
--fastx_revcomp command will reverse-complements sequences.
--eeout When using --fastq_filter, --fastx_filter or --fastq_mergepairs, include
the number of expected errors (ee) in the sequence header of FASTQ and
FASTA output files. This option is a synonym of the --fastq_eeout option.
Use the --xee option to remove this information from headers.
--eetabbedout filename
When specified with the --fastq_mergepairs command, write statistics with
expected errors of each merged read to the given file. The file is a tab
separated file with four columns: The number of errors expected in the
forward read, the number of expected errors in the reverse read, the
number of observed errors in the forward read, and the number of observed
errors in the reverse read. The observed number of errors are the number
of differences in the overlap region of the merged sequence relative to
each of the reads in the pair.
--fasta2fastq filename
Add a fake nucleotide quality score to the sequences in the given FASTA
file and write them to the FASTQ file specified with the --fastqout
option. The quality score may be adjusted using the --fastq_qmaxout option
(default 41). The --fastq_asciiout option may be used to adjust the FASTQ
output quality ASCII base character (default 33).
--fastaout filename
When using --fastq_filter, --fastq_mergepairs or --fastx_filter, write to
the given FASTA-formatted file the sequences passing the filter, or the
merged sequences.
--fastaout_rev filename
When using --fastq_filter, or --fastx_filter, write to the given FASTA-
formatted file the reverse reads passing the filter.
--fastaout_notmerged_fwd filename
When using --fastq_mergepairs, write forward reads not merged to the
specified FASTA file.
--fastaout_notmerged_rev filename
When using --fastq_mergepairs, write reverse reads not merged to the
specified FASTA file.
--fastaout_discarded filename
Write sequences that do not pass the filter of the --fastq_filter or
--fastx_filter command to the given FASTA-formatted file.
--fastaout_discarded_rev filename
Write reverse reads that do not pass the filter of the --fastq_filter or
--fastx_filter command to the given FASTA-formatted file.
--fastq_allowmergestagger
When using --fastq_mergepairs, allow merging of staggered read pairs.
Staggered pairs are pairs where the 3' end of the reverse read has an
overhang to the left of the 5' end of the forward read. This situation can
occur when a very short fragment is sequenced. The 3' overhang of the
reverse read is not included in the merged sequence. The opposite option
is the --fastq_nostagger option. The default is to discard staggered
pairs.
--fastq_ascii positive integer
Define the ASCII character number used as the basis for the FASTQ quality
score. The default is 33, which is used by the Sanger / Illumina 1.8+
FASTQ format (phred+33). The value 64 is used by the Solexa, Illumina 1.3+
and Illumina 1.5+ formats (phred+64). Only 33 and 64 are valid arguments.
--fastq_asciiout positive integer
When using --fastq_convert, --sff_convert or --fasta2fastq, define the
ASCII character number used as the basis for the FASTQ quality score when
writing FASTQ output files. The default is 33. Only 33 and 64 are valid
arguments.
--fastq_chars filename
Summarize the composition of sequence and quality strings contained in the
input FASTQ file. For each of the four DNA letters, --fastq_chars gives
the number of occurrences of the letter, its relative frequency and the
length of the longest run of that letter. For each character present in
the quality strings, --fastq_chars gives the ASCII value of the character,
its relative frequency, and the number of times a k-mer of that character
appears at the end of quality strings. The length of the k-mer can be set
using --fastq_tail (4 by default). The command --fastq_chars tries to
automatically detect the quality encoding (Solexa, Illumina 1.3+, Illumina
1.5+ or Illumina 1.8+/Sanger) by analyzing the range of observed quality
score values. In case of success, --fastq_chars suggests values for the
--fastq_ascii (33 or 64), --fastq_qmin and --fastq_qmax options to be used
with the other commands that require a FASTQ input file.
--fastq_convert filename
Convert between the different variants of the FASTQ file format. The
quality encoding of the input file must be specified with the
--fastq_ascii option (either 33 or 64, the default is 33), and the output
quality encoding must be specified with the --fastq_asciiout option
(default 33). The minimum and maximum output quality scores may be limited
using the --fastq_qminout and --fastq_qmaxout options. The output file is
specified with the --fastqout option.
--fastq_eeout
When using --fastq_filter, --fastx_filter or --fastq_mergepairs, include
the number of expected errors (ee) in the sequence header of FASTQ and
FASTA files. This option is a synonym of the --eeout option. Use the --xee
option to remove this information from headers.
--fastq_eestats filename
Analyze a FASTQ file and report statistics on the distributions of quality
scores, error probabilities and expected accumulated errors. The report, a
table of 21 tab-separated columns, is written to the file specified with
the --output option. The first column corresponds to the position in the
reads (Pos). The second and third columns correspond to the number of
reads (Reads) and percentage of reads (PctRecs) that include this
position. The remaining columns include information about the distribution
of quality scores in this position (Q), error probabilities in this
position (Pe), and finally the expected number of accumulated errors from
the beginning of the reads and until the current position (EE). For each
of the Q, Pe and EE distributions, the following statistics are included:
minimum value (Min), lower quartile (Low), median (Med), mean (Mean),
upper quartile (Hi), and maximum value (Max). The quality encoding and the
range of quality values may be specified with --fastq_ascii --fastq_qmin
and --fastq_qmax.
--fastq_eestats2 filename
Analyze the specified FASTQ file and report statistics on the number of
sequences that would be retained at a combination of selected cutoffs for
length truncation and maximum expected errors, that could potentially be
used as arguments to the --fastq_trunclen and --fastq_maxee options to the
--fastq_filter command. The result, a table of two or more columns, is
written to the file specified with the --output option. There is a line
for each length truncation cutoff. The first column on each line contains
the selected truncation length, while the following columns contain the
number of sequences and, in parenthesis, the percentage of sequences that
would be retained at the selected EE levels. The truncation length
cutoffs may be specified with the --length_cutoffs option and requires a
list of three comma-separated integers indicating the shortest cutoff, the
longest cutoff, and the increment between cutoffs. The longest cutoff may
be specified with a star (*) which indicates that the limit is equal to
the longest sequence in the input file. The default setting is "50,*,50"
meaning that truncation lengths of 50, 100, 150 and so on up to the
longest sequence length should be used. The maximum expected error (EE)
cutoffs may be specified with the --ee_cutoffs option which requires a
comma-separated list of floating point numbers as its argument. The
default setting is "0.5,1.0,2.0" that indicates that expected error levels
of 0.5, 1.0 and 2.0 should be used.
--fastq_filter filename
Trim and/or filter sequences in the given FASTQ file. Similar to the
--fastx_filter command, but works only on FASTQ files. See --fastx_filter
for details.
--fastq_join filename
Join paired-end sequence reads into one sequence and add a gap between
them using a padding sequence. The sequences are not merged as with the
fastq_mergepairs command, but simply joined with a gap. The forward reads
are specified as the argument to this option and the reverse reads are
specified with the --reverse option. The resulting sequences consist of
the forward read, the padding sequence and the reverse complement of the
reverse read. The padding sequence is specified with the --join_padgap
option and the padding quality is specified with the --join_padgapq
option. The default padding sequence string is NNNNNNNN and the default
padding quality string is IIIIIIII, corresponding to a base quality score
of 40 (a very high quality score with error probability 0.0001). The
joined sequences are output to the file(s) specified with the --fastaout
or --fastqout options.
--fastq_maxdiffs positive integer
When using --fastq_mergepairs, specify the maximum number of non-matching
nucleotides allowed in the overlap region. That option has a strong
influence on the merging success rate. The default value is 10.
--fastq_maxdiffpct real
When using --fastq_mergepairs, specify the maximum percentage of non-
matching nucleotides allowed in the overlap region. The default value is
100.0%. There are other more sophisticated rules in the merging algorithm
that will discard read pairs with a high fraction of mismatches.
--fastq_maxee real
When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard
sequences with an expected error greater than the specified number (value
ranging from 0.0 to infinity). For a given sequence, the expected error is
the sum of error probabilities for all the positions in the sequence. In
practice, the expected error is greater than zero (error probabilities can
be small but not null), and at most equal to the length of the sequence
(when all positions have an error probability of 1.0).
--fastq_maxee_rate real
When using --fastq_filter or --fastx_filter, discard sequences with an
average expected error greater than the specified number (value ranging
from 0.0 to 1.0 included). For a given sequence, the average expected
error is the sum of error probabilities for all the positions in the
sequence, divided by the length of the sequence.
--fastq_maxlen positive integer
When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard
sequences with more than the specified number of bases.
--fastq_maxmergelen positive integer
When using --fastq_mergepairs, specify the maximum length of the merged
sequence. By default there is no limit.
--fastq_maxns positive integer
When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard
sequences with more than the specified number of N's.
--fastq_mergepairs filename
Merge paired-end sequence reads into one sequence. The forward reads are
specified as the argument to this option and the reverse reads are
specified with the --reverse option. Reads with the same index/position in
the forward and reverse files are considered to form a pair, even if their
labels are different. Thus, forward and reverse reads must appear in the
same order and total number in both files. A warning is emitted if the
forward and reverse files contain different numbers of reads. The merged
sequences are written to the file(s) specified with the --fastaout or
--fastqout options. The non-merged reads can be output to the files
specified with the --fastaout_notmerged_fwd, --fastaout_notmerged_rev,
--fastqout_notmerged_fwd and --fastqout_notmerged_rev options. Statistics
may be output to the file specified with the --eetabbedout option.
Sequences are truncated as specified with the --fastq_truncqual option to
remove low-quality bases in the 3' end. Sequences shorter than specified
with --fastq_minlen (after truncation) are discarded (1 by default).
Sequences with too many ambiguous bases (N's), as specified with the
--fastq_maxns are also discarded (no limit by default). Staggered reads
are not merged unless the --fastq_allowmergestagger option is specified.
The minimum length of the overlap region between the reads may be
specified with the --fastq_minovlen option (at least 5, default 10). The
overlap region may not include more mismatches than specified with the
--fastq_maxdiffs option (10 by default) or a higher percentage of
mismatches than specified with the --fastq_maxdiffpct option (100.0% by
default), otherwise the read pair is discarded. Additional rules will
avoid merging of reads that cannot be aligned reliably and unambiguously.
The minimum and maximum length of the merged sequence may be specified
with the --fastq_minmergelen and --fastq_maxmergelen options,
respectively. The quality value limits for output files may be specified
with the --fastq_qminout and --fastq_qmaxout options, but they apply only
to the merged region. Other relevant options are: --fastq_ascii,
--fastq_maxee, --fastq_nostagger, --fastq_qmax, --fastq_qmin, and
--label_suffix.
--fastq_minlen positive integer
When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard
sequences with less than the specified number of bases (default 1).
--fastq_minmergelen positive integer
When using --fastq_mergepairs, specify the minimum length of the merged
sequence. The default is 1.
--fastq_minovlen positive integer
When using --fastq_mergepairs, specify the minimum overlap between the
merged reads. The default is 10. Must be at least 5.
--fastq_nostagger
When using --fastq_mergepairs, forbid the merging of staggered read pairs.
This is the default behaviour of --fastq_mergepairs. To change that
behaviour, see the --fastq_allowmergestagger option.
--fastq_qmax positive integer
Specify the maximum quality score accepted when reading FASTQ files. The
default is 41, which is usual for recent Sanger/Illumina 1.8+ files.
--fastq_qmaxout positive integer
When using --fastq_mergepairs, --fastq_convert, --sff_convert or
--fasta2fastq, specify the maximum quality score used when writing FASTQ
files. For the --fasta2fastq command, the value specified here is the fake
quality score used for the FASTQ output file. The default is 41, which is
usual for recent Sanger/Illumina 1.8+ files. Older formats may use a
maximum quality score of 40. The limit only applies to the merged region
when using --fastq_mergepairs.
--fastq_qmin positive integer
Specify the minimum quality score accepted for FASTQ files. The default is
0, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may
use scores between -5 and 2.
--fastq_qminout positive integer
When using --fastq_mergepairs, --fastq_convert or --sff_convert, specify
the minimum quality score used when writing FASTQ files. The default is 0,
which is usual for Sanger/Illumina 1.8+ files. Older versions of the
format may use scores between -5 and 2. The limit applies only to the
merged region when using --fastq_mergepairs.
--fastq_stats filename
Analyze a FASTQ file and report the number of reads it contains. The
quality encoding and the range of quality values may be specified with
--fastq_ascii --fastq_qmin and --fastq_qmax. That command requires the
--log option and outputs the following detailed statistics on read length,
quality score, length vs. quality distributions, and length / quality
filtering:
Read length distribution:
1. L: read length.
2. N: number of reads.
3. Pct: fraction of reads with this length.
4: AccPct: fraction of reads with this length or longer.
Quality score distribution:
1. ASCII: character encoding the quality score.
2. Q: Phred quality score.
3. Pe: probability of error associated with the quality score.
4. N: number of bases with this quality score.
5. Pct: fraction of bases with this quality score.
6: AccPct: fraction of bases with this quality score or higher.
Length vs. quality distribution:
1. L: position in reads (starting from position 2).
2. PctRecs: fraction of reads with at least this length.
3. AvgQ: average quality score over all reads up to this position.
4. P(AvgQ): error probability corresponding to AvgQ.
5. AvgP: average error probability.
6: AvgEE: average expected error over all reads up to this
position.
7: Rate: growth rate of AvgEE between this position and position -
1.
8: RatePct: Rate (as explained above) expressed as a percentage.
Effect of expected error and length filtering:
The first column indicates read lengths (L). The next four columns
indicate the number of reads that would be retained by the
--fastq_filter command if the reads were truncated at length L
(option --fastq_trunclen L) and filtered to have a maximum expected
error of 1.0, 0.5, 0.25 or 0.1 (with the option --fastq_maxee
float). The last four columns indicate the fraction of reads that
would be retained by the --fastq_filter command using the same
length and maximum expected error parameters.
Effect of minimum quality and length filtering:
The first column indicates read lengths (Len). The next four
columns indicate the fraction of reads that would be retained by
the --fastq_filter command if the reads were truncated at length
Len (option --fastq_trunclen Len) or at the first position with a
quality Q below 5, 10, 15 or 20 (option --fastq_truncqual Q).
--fastq_stripleft positive integer
When using --fastq_filter or --fastx_filter, strip the specified number of
bases from the left end of the reads.
--fastq_stripright positive integer
When using --fastq_filter or --fastx_filter, strip the specified number of
bases from the right end of the reads.
--fastq_tail positive integer
When using --fastq_chars, count the number of times a series of characters
of length k appears at the end of quality strings. By default, k = 4.
--fastq_truncee real
When using --fastq_filter or --fastx_filter, truncate sequences so that
their total expected error is not higher than the specified value.
--fastq_trunclen positive integer
When using --fastq_filter or --fastx_filter, truncate sequences to the
specified length. Shorter sequences are discarded.
--fastq_trunclen_keep positive integer
When using --fastq_filter or --fastx_filter, truncate sequences to the
specified length. Shorter sequences are not discarded.
--fastq_truncqual positive integer
When using --fastq_filter or --fastx_filter, truncate sequences starting
from the first base with the specified base quality score value or lower.
--fastqout filename
When using --fastq_filter, --fastq_mergepairs, --fastx_filter or
--fasta2fastq, write to the given FASTQ-formatted file the sequences
passing the filter, or the merged or converted sequences.
--fastqout_rev filename
When using --fastq_filter or --fastx_filter, write to the given FASTQ-
formatted file the reverse reads passing the filter.
--fastqout_discarded filename
When using --fastq_filter or --fastx_filter, write sequences that do not
pass the filter to the given FASTQ-formatted file.
--fastqout_discarded_rev filename
When using --fastq_filter or --fastx_filter, write reverse reads that do
not pass the filter to the given FASTQ-formatted file.
--fastqout_notmerged_fwd filename
When using --fastq_mergepairs, write forward reads not merged to the
specified FASTQ file.
--fastqout_notmerged_rev filename
When using --fastq_mergepairs, write reverse reads not merged to the
specified FASTQ file.
--fastx_filter filename
Trim and/or filter the sequences in the given FASTA or FASTQ file and
output the remaining sequences to the FASTQ file specified with the
--fastqout option and/or to the FASTA file specified with the --fastaout
option. Discarded sequences are written to the files specified with the
--fastaout_discarded and --fastqout_discarded options. The input format
(FASTA or FASTQ) is automatically detected. If the input consists of
paired sequences, an input file with reverse reads may be specified with
the --reverse option, and corresponding output will be written to the
files specified with the --fastqout_rev, --fastaout_rev,
--fastqout_discarded_rev, and --fastaout_discarded_rev options. Output can
not be written to FASTQ files if the input is in FASTA format. The
sequences are first trimmed and then filtered based on the remaining
bases. Sequences may be trimmed using the options --fastq_stripleft,
--fastq_stripright, --fastq_truncee, --fastq_trunclen,
--fastq_trunclen_keep and --fastq_truncqual. The sequences may be filtered
using the options --fastq_maxee, --fastq_maxee_rate, --fastq_maxlen,
--fastq_maxns, --fastq_minlen (default 1), --fastq_trunclen, --maxsize,
and --minsize. Sequences not satisfying the requirements are discarded.
For pairs of sequences, both sequences in a pair must satisfy the
requirements, otherwise both are discarded. If no shortening or filtering
options are given, all sequences are written to the output files, possibly
after conversion from FASTQ to FASTA format. The --relabel option may be
used to relabel the output sequences. The --eeout option may be used to
output the expected number of errors in each sequence. After all sequences
have been processed, the number of kept and discarded sequences will be
shown, as well as how many of the kept sequences were trimmed. When the
input is in FASTA format, the following options are not accepted because
quality scores are not available: --eeout, --fastq_ascii, --fastq_eeout,
--fastq_maxee, --fastq_maxee_rate, --fastq_out, --fastq_qmax,
--fastq_qmin, --fastq_truncee, --fastq_truncqual, --fastqout_discarded,
--fastqout_discarded_rev, --fastqout_rev.
--fastx_revcomp filename
Reverse-complement the sequences in the given FASTA or FASTQ file to a
file specified with the --fastaout and/or --fastqout options. If the input
file is in FASTA format, the output can not be written back to a FASTQ
file due to missing base quality scores.
--join_padgap string
When running --fastq_join, use the string as a sequence padding string.
The default is NNNNNNNN (8 N's).
--join_padgapq string
When running --fastq_join, use the string as a quality padding string. The
default is a string of I's equal in length to the sequence padding string.
The letter I corresponds to a base quality score of 40 indicating a very
high quality base with error probability of 0.0001.
--maxsize positive integer
When using --fastq_filter or --fastx_filter, discard sequences with an
abundance higher than the specified value.
--minsize positive integer
When using --fastq_filter or --fastx_filter, discard sequences with an
abundance lower than the specified value.
--output filename
When using --fastq_eestats or --fastq_eestats2, write tabulated results to
filename. See --fastq_eestats's and --fastq_eestats2's documentation for a
complete description of the table.
--relabel_keep
When using --relabel, keep the old identifier in the header after a space.
--relabel string
Please see the description of the same option under Chimera detection for
details.
--relabel_md5
Please see the description of the same option under Chimera detection for
details.
--relabel_self
Please see the description of the same option under Chimera detection for
details.
--relabel_sha1
Please see the description of the same option under Chimera detection for
details.
--reverse filename
When using --fastq_filter, --fastx_filter, --fastq_mergepairs or
--fastq_join, specify the FASTQ file containing containing the reverse
reads.
--sff_convert filename
Convert the given SFF file to FASTQ. The FASTQ output file is specified
with the --fastqout option. The sequence may be clipped as specified in
the SFF file if the option --sff_clip is specified, otherwise no clipping
occurs. Bases that would have been clipped are converted to lower case,
while the rest is in upper case. The output quality encoding may be
specified with the --fastq_asciiout option (default 33). The minimum and
maximum output quality scores may be limited using the --fastq_qminout and
--fastq_qmaxout options.
--sff_clip
Specifies that the sequences converted by the --sff_convert command should
be clipped in both ends as indicated in the SFF file. By default no
clipping is performed.
--xsize Strip abundance information from the headers when writing the output file.
--xee Strip information about expected errors (ee) from the output file headers.
This information is added by the --fastq_eeout and --eeout options.
Masking options:
An input sequence can be composed of lower- or uppercase letters. When soft masking
is specified, lower case letters are treated as symbols that should be masked.
Otherwise the case of the input sequences is ignored.
Masking is performed by the commands for chimera detection (uchime_denovo,
uchime_ref), clustering (cluster_fast, cluster_smallmem, cluster_size), masking
(maskfasta, fastx_mask), pairwise alignment (allpairs_global) and searching
(search_exact, usearch_global).
Masking is usually specified with the --qmask option, while the --dbmask option is
used for the database sequences specified with the --db option with the
--usearch_global, --search_exact and --uchime_ref commands.
The argument to the --qmask and --dbmask option may be none, soft or dust. If the
argument is none, the no masking is performed. If the argument is soft the lower
case symbols are masked. Finally, if the argument is dust, the sequence is masked
using the DUST algorithm by Tatusov and Lipman to mask low-complexity regions.
If the --hardmask option is specified, all masked regions are converted to N's,
otherwise masked regions are indicated by lower case letters.
If any sequence is masked, the masked version of the sequence (with lower case
letters or N's) is used in all output files. Otherwise the sequence is unmodified.
The exception is the sequences in the output file specified with the --uchimealns
option, where the input sequences are converted to upper case first and lower case
letters indicate disagreement between the aligned sequences.
The --qmask option (or --dbmask for database sequences) may be combined with the
--hardmask option. The results of using the none, dust or soft argument to --qmask
or --dbmask are presented below, assuming each input sequence contains both lower
and uppercase symbols.
Results if the --hardmask option is off (default):
none: no masking, all symbols used, no change
dust: masked symbols lowercased, rest uppercased
soft: lowercase symbols masked, no case changes
Results if the --hardmask option is on:
none: no masking, all symbols used, no change
dust: masked symbols changed to Ns, rest unchanged
soft: lowercase symbols masked and changed to Ns
When a sequence region is masked, words in the region are not included in the
indices used in the heuristic search algorithm. In all other aspects, the region is
treated as other regions.
Regions in sequences that are hardmasked (with N's) have a zero alignment score and
do not contribute to an alignment.
--fastaout filename
Write the masked sequences to filename, in fasta format. Applies only to
the --fastx_mask command.
--fastqout filename
Write the masked sequences to filename, in fastq format. Applies only to
the --fastx_mask command.
--fastx_mask filename
Mask regions in sequences contained in the specified fasta or fastq file.
The default is to mask using DUST (use --qmask to modify that behavior).
The output files are specified with the --fastaout and --fastqout options.
The minimum and maximum percentage of unmasked residues may be specified
with the --min_unmasked_pct and --max_unmasked_pct options, respectively.
--hardmask
Symbols in masked regions are replaced by N's. The default is to replace
the masked regions by lower case letters.
--maskfasta filename
Mask regions in sequences contained in the fasta file filename. The
default is to mask using dust (use --qmask to modify that behavior). The
output file is specified with the --output option. This command is
depreciated, please use --fastx_mask instead.
--max_unmasked_pct real
Discard sequences with more than the specified maximum percentage of
unmasked residues. Works only with --fastx_mask.
--min_unmasked_pct real
Discard sequences with less than the specified minimum percentage of
unmasked residues. Works only with --fastx_mask.
--output filename
Write the masked sequences to filename, in fasta format. Applies only to
the --mask_fasta command.
--qmask none|dust|soft
If the argument is dust, mask regions in sequences using the DUST
algorithm that detects simple repeats and low-complexity regions. This is
the default. If the argument is soft, mask the lower case letters in the
input sequence. If the argument is none, do not mask.
Orienting options:
The --orient command can be used to orient the sequences in a given file in either
the forward or the reverse complementary direction based on a reference database
specified with the --db option. The two strands of each input sequence are compared
to the reference database using nucleotide words. If one of the strands share many
more words with at least one sequence in the database than the other, that strand
is chosen. The correctly oriented sequences may be written to a FASTA file
specified with the --fastaout, and to a FASTQ file specified with the --fastqout
option (as long as the input was also in FASTA format). If the result is uncertain,
because the number of matching words is too similar, the original sequence is
written to the file specified with the --notmatched option. The results may also be
written to a tab-delimited text file specified with the --tabbedout option. This
file will contain the query label, the direction (+, - or ?), the number of
matching words on the forward strand, and the number of matching words on the
reverse complementary strand. By default, a word length of 12 is used for this
command. The word length may be adjusted using the --wordlength option. There has
to be at least 4 times as many matches on one strand than the other for a strand to
be selected. In addition to the common options, the following options may also be
specified for this command: --dbmask, --qmask, --relabel, --relabel_keep,
--relabel_md5, --relabel_self, --relabel_sha1, --sizein, and --sizeout.
--db filename
Read the reference database from the given file. It may be in FASTA, FASTQ
or UDB format. If an UDB file is used it should have been created with a
wordlength of 12.
--fastaout filename
Write the correctly oriented sequences to filename, in fasta format.
--fastqout filename
Write the correctly oriented sequences to filename, in fastq format.
--notmatched filename
Write the sequences with undetermined direction to filename, in the
original format.
--orient filename
Orient the sequences in the given file.
--tabbedout filename
Write the resuls to a tab-delimited text file with the specified filename.
This file will contain the query label, the direction (+, - or ?), the
number of matching words on the forward strand, and the number of matching
words on the reverse complementary strand.
Pairwise alignment options:
The results of the n * (n-1) / 2 pairwise alignments are written to the result
files specified with --alnout, --blast6out, --fastapairs --matched, --notmatched,
--qsegout, --samout, --tsegout, --uc or --userout (see Searching section below).
Specify either the --acceptall option to output all pairwise alignments, or specify
an identity level with --id to discard weak alignments. Most other accept/reject
options (see Searching options below) may also be used. Sequences are aligned on
their plus strand only. Masking is performed as usual and specified with --qmask
and --hardmask.
--acceptall
Write the results of all alignments to output files. This option overrides
all other accept/reject options (including --id).
--allpairs_global filename
Perform optimal global pairwise alignments of the fasta sequences
contained in filename. Each sequence is compared to all sequencs that come
after it in the file, resulting in a total of n * (n-1) / 2 pairwise
alignments, where n is the total number of sequences. This command is
multi-threaded.
--id real
Reject the sequence match if the pairwise identity is lower than real
(value ranging from 0.0 to 1.0 included).
--threads positive integer
Number of computation threads to use (1 to 1024). The number of threads
should be lesser or equal to the number of available CPU cores. The
default is to use all available resources and to launch one thread per
logical core.
--uc filename
Output pairwise alignment results in filename using a tab-separated
uclust-like format with 10 columns. Each sequence is compared to all other
sequences, and all hits (--acceptall) or only some hits (--id float) are
reported, with one pairwise comparison per line:
1. Record type, always set to 'H'.
2. Ordinal number of the target sequence (based on input order,
starting from zero).
3. Sequence length.
4. Percentage of similarity with the target sequence.
5. Match orientation, always set to '+'.
6. Not used, always set to zero.
7. Not used, always set to zero.
8. Compact representation of the pairwise alignment using the
CIGAR format (Compact Idiosyncratic Gapped Alignment Report): M
(match/mismatch), D (deletion) and I (insertion). The equal
sign '=' indicates that the query is identical to the centroid
sequence.
9. Label of the query sequence.
10. Label of the target sequence.
Restriction site cutting options:
The input sequences in the file specified with the --cut command are cut into
fragments at all restriction sites matching the pattern given with the
--cut_pattern option. The fragments on the forward strand are written to the file
specified with the --fastaout file and the fragments on the reverse strand are
written to the file specified with the --fastaout_rev option. Input sequences that
do not match are written to the file specified with the option
--fastaout_discarded, and their reverse complement are also written to the file
specified with the --fastaout_discarded_rev option. The relabel options (--relabel,
--relabel_self, --relabel_keep, --relabel_md5, and --relabel_sha1) may be used to
relabel the output sequences).
--cut filename
Specify the input file with sequences in FASTA format.
--cut_pattern string
Specify the restriction site cutting pattern and positions. The pattern is
a string of lower- or uppercase letters specifying the nucleotides that
must match, and may include ambiguous nucleotide symbols. The special
characters "^" (circumflex) and "_" (underscore) are used to indicate the
cutting position on the forward and reverse strand, respectively. For
example, the pattern "G^AATT_C" is the pattern for the EcoRI restriction
site. For such palindromic patterns (identical to its reverse complement)
the command will output all possible fragments on both strands. For non-
palindromic sites, it may be necessary to run the command also on the
reverse complemented input sequences. Exactly one cutting site on each
strand must be indicated.
--fastaout filename
Specify the output file for the resulting fragments on the forward strand.
--fastaout_rev filename
Specify the output file for the resulting fragments on the reverse strand.
--fastaout_discarded filename
Specify the output file for the non-matching sequences.
--fastaout_discarded_rev filename
Specify the output file for the non-matching seqeunces, reverse
complemented.
Searching options:
--alnout filename
Write pairwise global alignments to filename using a human-readable
format. Use --rowlen to modify alignment length. Output order may vary
when using multiple threads.
--biomout filename
Write search results to an OTU table in the biom version 1.0 file format.
The query file contains the samples, while the database file contains the
OTUs. Sample and OTU identifiers are extracted from the header of these
sequences. See the --biomout option in the Clustering section for further
details.
--blast6out filename
Write search results to filename using a blast-like tab-separated format
of twelve fields (listed below), with one line per query-target matching
(or lack of matching if --output_no_hits is used). Warning, vsearch uses
global pairwise alignments, not blast's seed-and-extend algorithm.
Therefore, some common blast output values (alignment start and end,
evalue, bit score) are reported differently. Output order may vary when
using multiple threads. A similar output can be obtain with --userout
filename and --userfields
query+target+id+alnlen+mism+opens+qlo+qhi+tlo+thi+evalue+bits. A complete
list and description is available in the section 'Userfields' of this
manual.
1. query: query label.
2. target: target (database sequence) label. The field is set to
'*' if there is no alignment.
3. id: percentage of identity (real value ranging from 0.0 to
100.0). The percentage identity is defined as 100 * (matching
columns) / (alignment length - terminal gaps). See fields id0
to id4 for other definitions.
4. alnlen: length of the query-target alignment (number of
columns). The field is set to 0 if there is no alignment.
5. mism: number of mismatches in the alignment (zero or positive
integer value).
6. opens: number of columns containing a gap opening (zero or
positive integer value).
7. qlo: first nucleotide of the query aligned with the target.
Always equal to 1 if there is an alignment, 0 otherwise (see
qilo to ignore initial gaps).
8. qhi: last nucleotide of the query aligned with the target.
Always equal to the length of the pairwise alignment, 0
otherwise (see qihi to ignore terminal gaps).
9. tlo: first nucleotide of the target aligned with the query.
Always equal to 1 if there is an alignment, 0 otherwise (see
tilo to ignore initial gaps).
10. thi: last nucleotide of the target aligned with the query.
Always equal to the length of the pairwise alignment, 0
otherwise (see tihi to ignore terminal gaps).
11. evalue: expectancy-value (not computed for nucleotide
alignments). Always set to -1.
12. bits: bit score (not computed for nucleotide alignments).
Always set to 0.
--db filename
Compare query sequences (specified with --usearch_global) to the fasta-
formatted target sequences contained in filename, using global pairwise
alignment. Alternatively, the name of a preformatted UDB database created
using the makeudb_usearch command (see below) may be specified.
--dbmask none|dust|soft
Mask regions in the target database sequences using the dust method or the
soft method, or do not mask (none). Warning, when using soft masking
search commands become case sensitive. The default is to mask using dust.
--dbmatched filename
Write database target sequences matching at least one query sequence to
filename, in fasta format. If the option --sizeout is used, the number of
queries that matched each target sequence is indicated using the pattern
";size=integer;".
--dbnotmatched filename
Write database target sequences not matching query sequences to filename,
in fasta format.
--fastapairs filename
Write pairwise alignments of query and target sequences to filename, in
fasta format.
--fulldp Dummy option for compatibility with usearch. To maximize search
sensitivity, vsearch uses a 8-way 16-bit SIMD vectorized full dynamic
programming algorithm (Needleman-Wunsch), whether or not --fulldp is
specified.
--gapext string
Set penalties for a gap extension. See --gapopen for a complete
description of the penalty declaration system. The default is to
initialize the six gap extending penalties using a penalty of 2 for
extending internal gaps and a penalty of 1 for extending terminal gaps, in
both query and target sequences (i.e. 2I/1E).
--gapopen string
Set penalties for a gap opening. A gap opening can occur in six different
contexts: in the query (Q) or in the target (T) sequence, at the left (L)
or right (R) extremity of the sequence, or inside the sequence (I).
Sequence symbols (Q and T) can be combined with location symbols (L, I,
and R), and numerical values to declare penalties for all possible
contexts: aQL/bQI/cQR/dTL/eTI/fTR, where abcdef are zero or positive
integers, and '/' is used as a separator.
To simplify declarations, the location symbols (L, I, and R) can be
combined, the symbol (E) can be used to treat both extremities (L and R)
equally, and the symbols Q and T can be omitted to treat query and target
sequences equally. For instance, the default is to declare a penalty of 20
for opening internal gaps and a penalty of 2 for opening terminal gaps
(left or right), in both query and target sequences (i.e. 20I/2E). If only
a numerical value is given, without any sequence or location symbol, then
the penalty applies to all gap openings. To forbid gap-opening, an
infinite penalty value can be declared with the symbol '*'. To use vsearch
as a semi-global aligner, a null-penalty can be applied to the left (L) or
right (R) gaps.
vsearch always initializes the six gap opening penalties using the default
parameters (20I/2E). The user is then free to declare only the values
he/she wants to modify. The string is scanned from left to right, accepted
symbols are (0123456789/LIREQT*), and later values override previous
values.
Please note that vsearch, in contrast to usearch, only allows integer gap
penalties. Because the lowest gap penalties are 0.5 by default in usearch,
all default scores and gap penalties in vsearch have been doubled to
maintain equivalent penalties and to produce identical alignments.
--hardmask
Mask sequence regions by replacing them with Ns instead of setting them to
lower case as is the default. For more information, please see the Masking
section.
--id real
Reject the sequence match if the pairwise identity is lower than real
(value ranging from 0.0 to 1.0 included). The search process sorts target
sequences by decreasing number of k-mers they have in common with the
query sequence, using that information as a proxy for sequence similarity.
That efficient pre-filtering also prevents pairwise alignments with weakly
matching targets, as there needs to be at least 6 shared k-mers to start
the pairwise alignment, and at least one out of every 16 k-mers from the
query needs to match the target. Consequently, using values lower than
--id 0.5 is not likely to capture more weakly matching targets. The
pairwise identity is by default defined as the number of (matching
columns) / (alignment length - terminal gaps). That definition can be
modified by --iddef.
--iddef 0|1|2|3|4
Change the pairwise identity definition used in --id. Values accepted are:
0. CD-HIT definition: (matching columns) / (shortest sequence
length).
1. edit distance: (matching columns) / (alignment length).
2. edit distance excluding terminal gaps (default definition for
--id).
3. Marine Biological Lab definition counting each gap opening
(internal or terminal) as a single mismatch, whether or not the
gap was extended: 1.0 - [(mismatches + gap openings)/(longest
sequence length)]
4. BLAST definition, equivalent to --iddef 1 for global pairwise
alignments.
The option --userfields accepts the fields id0 to id4, in addition to the
field id, to report the pairwise identity values corresponding to the
different definitions.
--idprefix positive integer
Reject the sequence match if the first integer nucleotides of the target
do not match the query.
--idsuffix positive integer
Reject the sequence match if the last integer nucleotides of the target do
not match the query.
--lca_cutoff real
Adjust the fraction of matching hits required for the last common ancestor
(LCA) output with the --lcaout option during searches. The default value
is 1.0 which requires all hits to match at each taxonomic rank for that
rank to be included. If a lower cutoff value is used, e.g. 0.95, a small
fraction of non-matching hits are allowed while that rank will still be
reported. The argument to this option must be larger than 0.5, but not
larger than 1.0.
--lcaout filename
Output last common ancestor (LCA) information about the hits of each query
to a text file in a tab-separated format. The first column contains the
query id, while the second column contains the taxonomic information. The
headers of the sequences in the database must contain taxonomic
information in the same format as used with the --sintax command, e.g.
"tax=k:Archaea,p:Euryarchaeota,c:Halobacteria". Only the initial parts of
the taxonomy that are common to a large fraction of the hits of each query
will be output. It is necessary to set the --maxaccepts option to a value
different from 1 for this information to be useful. The --top_hits_only
option may also be useful. The fraction of matching hits required may be
adjusted by the --lca_cutoff option (default 1.0).
--leftjust
Reject the sequence match if the pairwise alignment begins with gaps.
--match integer
Score assigned to a match (i.e. identical nucleotides) in the pairwise
alignment. The default value is 2.
--matched filename
Write query sequences matching database target sequences to filename, in
fasta format.
--maxaccepts positive integer
Maximum number of hits to accept before stopping the search. The default
value is 1. This option works in pair with --maxrejects. The search
process sorts target sequences by decreasing number of k-mers they have in
common with the query sequence, using that information as a proxy for
sequence similarity. After pairwise alignments, if the first target
sequence passes the acceptation criteria, it is accepted as best hit and
the search process stops for that query. If --maxaccepts is set to a
higher value, more hits are accepted. If --maxaccepts and --maxrejects are
both set to 0, the complete database is searched.
--maxdiffs positive integer
Reject the sequence match if the alignment contains at least integer
substitutions, insertions or deletions.
--maxgaps positive integer
Reject the sequence match if the alignment contains at least integer
insertions or deletions.
--maxhits non-negative integer
Maximum number of hits to show once the search is terminated (hits are
sorted by decreasing identity). Unlimited by default or if the argument it
zero. This option applies to --alnout, --blast6out, --fastapairs,
--samout, --uc, or --userout output files.
--maxid real
Reject the sequence match if the percentage of identity between the two
sequences is greater than real.
--maxqsize positive integer
Reject query sequences with an abundance greater than integer.
--maxqt real
Reject if the query/target sequence length ratio is greater than real.
--maxrejects positive integer
Maximum number of non-matching target sequences to consider before
stopping the search. The default value is 32. This option works in pair
with --maxaccepts. The search process sorts target sequences by decreasing
number of k-mers they have in common with the query sequence, using that
information as a proxy for sequence similarity. After pairwise alignments,
if none of the first 32 examined target sequences pass the acceptation
criteria, the search process stops for that query (no hit). If
--maxrejects is set to a higher value, more target sequences are
considered. If --maxaccepts and --maxrejects are both set to 0, the
complete database is searched.
--maxsizeratio real
Reject if the query/target abundance ratio is greater than real.
--maxsl real
Reject if the shorter/longer sequence length ratio is greater than real.
--maxsubs positive integer
Reject the sequence match if the pairwise alignment contains more than
integer substitutions.
--mid real
Reject the sequence match if the percentage of identity is lower than real
(ignoring all gaps, internal and terminal).
--mincols positive integer
Reject the sequence match if the alignment length is shorter than integer.
--minqt real
Reject if the query/target sequence length ratio is lower than real.
--minsizeratio real
Reject if the query/target abundance ratio is lower than real.
--minsl real
Reject if the shorter/longer sequence length ratio is lower than real.
--mintsize positive integer
Reject target sequences with an abundance lower than integer.
--minwordmatches non-negative integer
Minimum number of word matches required for a sequence to be considered
further. Default value is 12 for the default word length 8. For word
lengths 3-15, the default minimum word matches are 18, 17, 16, 15, 14, 12,
11, 10, 9, 8, 7, 5 and 3, respectively. If the query sequence has fewer
unique words than the number specified, all words in the query must match.
If the argument is 0, no word matches are required.
--mismatch integer
Score assigned to a mismatch (i.e. different nucleotides) in the pairwise
alignment. The default value is -4.
--mothur_shared_out filename
Write search results to an OTU table in the mothur 'shared' tab-separated
plain text file format. The query file contains the samples, while the
database file contains the OTUs. Sample and OTU identifiers are extracted
from the header of these sequences. See the --otutabout option in the
Clustering section for further details.
--notmatched filename
Write query sequences not matching database target sequences to filename,
in fasta format.
--otutabout filename
Write search results to an OTU table in the classic tab-separated plain
text format. The query file contains the samples, while the database file
contains the OTUs. Sample and OTU identifiers are extracted from the
header of these sequences. See the --mothur_shared_out option in the
Clustering section for further details.
--output_no_hits
Write both matching and non-matching queries to --alnout, --blast6out,
--samout or --userout output files. Non-matching queries are labelled 'No
hits' in --alnout files.
--pattern string
This option is ignored. It is provided for compatibility with usearch.
--qmask none|dust|soft
Mask regions in the query sequences using the dust or the soft algorithms,
or do not mask (none). Warning, when using soft masking search commands
become case sensitive. The default is to mask using dust.
--qsegout filename
Write the aligned part of each query sequence to filename in FASTA format.
--query_cov real
Reject if the fraction of the query aligned to the target sequence is
lower than real (value ranging from 0.0 to 1.0 included). The query
coverage is computed as (matches + mismatches) / query sequence length.
Internal or terminal gaps are not taken into account.
--rightjust
Reject the sequence match if the pairwise alignment ends with gaps.
--rowlen positive integer
Width of alignment lines in --alnout output. The default value is 64. Set
to 0 to eliminate wrapping.
--samheader
Include header lines to the SAM file when --samout is specified. The
header includes lines starting with @HD, @SQ and @PG, but no @RG lines
(see <https://github.com/samtools/hts-specs>). By default no header line
is written.
--samout filename
Write alignment results to filename using the SAM format (a tab-separated
text file). When using the --samheader option, the SAM file starts with
header lines. Each non-header line is a SAM record, which represents
either a query-target alignment or the absence of match for a query
(output order may vary when using multiple threads). Each record contains
11 mandatory fields and optional fields (see
<https://github.com/samtools/hts-specs> for a complete description of the
format):
1. query sequence label.
2. combination of bitwise flags. Possible values are: 0 (top hit),
4 (no hit), 16 (reverse-complemented hit), 256 (secondary hit,
i.e. all hits except the top hit).
3. target sequence label.
4. first position of a target aligned with the query (always 1 for
global pairwise alignments, 0 if there is no match).
5. mapping quality (ignored, always set to '*').
6. CIGAR string (set to '*' if there is no match).
7. name of the target sequence matching with the next read of the
query (for mate reads only, ignored and always set to '*').
8. position of the primary alignment of the next read of the query
(for mate reads only, ignored and always set to 0).
9. target sequence length (for multi-segment targets, ignored and
always set to 0).
10. query sequence (complete, not only the segment aligned to the
target as usearch does).
11. quality string (ignored, always set to '*').
Optional fields for query-target matches (number and order of fields may
vary):
12. AS:i:? alignment score (i.e. percentage of identity).
13. XN:i:? next best alignment score (always set to 0).
14. XM:i:? number of mismatches.
15. XO:i:? number of gap openings (excluding terminal gaps).
16. XG:i:? number of gap extensions (excluding terminal gaps).
17. NM:i:? edit distance to the target (sum of XM and XG).
18. MD:Z:? string for mismatching positions.
19. YT:Z:UU string representing the alignment type.
--search_exact filename
Search for exact full-length matches to the query sequences contained in
filename in the database of target sequences (--db). Only 100% exact
matches are reported and this command is much faster than
--usearch_global. The --id, --maxaccepts and --maxrejects options are
ignored, but the rest of the searching options may be specified.
--self Reject the sequence match if the query and target labels are identical.
--selfid Reject the sequence match if the query and target sequences are strictly
identical.
--sizeout
Add abundance annotations to the output of the option --dbmatched (using
the pattern ';size=integer;'), to report the number of queries that
matched each target.
--strand plus|both
When searching for similar sequences, check the plus strand only (default)
or check both strands.
--target_cov real
Reject the sequence match if the fraction of the target sequence aligned
to the query sequence is lower than real. The target coverage is computed
as (matches + mismatches) / target sequence length. Internal or terminal
gaps are not taken into account.
--top_hits_only
Only the top hits with an equally high percentage of identity between the
query and database sequence sets are written to the output specified with
the options --lcaout, --alnout, --samout, --userout, --blast6out, --uc,
--fastapairs, --matched or --notmatched (but not --dbmatched and
--dbnotmatched). For each query, the top hit is the one presenting the
highest percentage of identity (see the --iddef option to change the way
identity is measured). For a given query, if several top hits present
exactly the same percentage of identity, the number of hits reported is
controlled by the --maxaccepts value (1 by default).
--tsegout filename
Write the aligned part of each target sequence to filename in FASTA
format.
--uc filename
Output searching results in filename using a tab-separated uclust-like
format with 10 columns. When using the --search_exact command, the table
layout is the same than with the --allpairs_global. When using the
--usearch_global command, the table present two different type of entries:
hit (H) or no hit (N). Each query sequence is compared to all other
sequences, and the best hit (--maxaccept 1) or several hits (--maxaccept >
1) are reported (H). Output order may vary when using multiple threads.
Column content varies with the type of entry (H or N):
1. Record type: H, or N ('hit' or 'no hit').
2. Ordinal number of the target sequence (based on input order,
starting from zero). Set to '*' for N.
3. Sequence length. Set to '*' for N.
4. Percentage of similarity with the target sequence. Set to '*'
for N.
5. Match orientation + or -. . Set to '.' for N.
6. Not used, always set to zero for H, or '*' for N.
7. Not used, always set to zero for H, or '*' for N.
8. Compact representation of the pairwise alignment using the
CIGAR format (Compact Idiosyncratic Gapped Alignment Report): M
(match/mismatch), D (deletion) and I (insertion). The equal
sign '=' indicates that the query is identical to the centroid
sequence. Set to '*' for N.
9. Label of the query sequence.
10. Label of the target centroid sequence. Set to '*' for N.
--uc_allhits
When using the --uc option, show all hits, not just the top hit for each
query.
--usearch_global filename
Compare target sequences (--db) to the fasta-formatted query sequences
contained in filename, using global pairwise alignment.
--userfields string
When using --userout, select and order the fields written to the output
file. Fields are separated by '+' (e.g. query+target+id). See the
'Userfields' section for a complete list of fields.
--userout filename
Write user-defined tab-separated output to filename. Select the fields
with the option --userfields. Output order may vary when using multiple
threads. If --userfields is empty or not present, filename is empty.
--weak_id real
Show hits with percentage of identity of at least real, without
terminating the search. A normal search stops as soon as enough hits are
found (as defined by --maxaccepts, --maxrejects, and --id). As --weak_id
reports weak hits that are not deduced from --maxaccepts, high --id values
can be used, hence preserving both speed and sensitivity. Logically, real
must be smaller than the value indicated by --id.
--wordlength positive integer
Length of words (i.e. k-mers) for database indexing. The range of possible
values goes from 3 to 15, but values near 8 or 9 are generally
recommended. Longer words may reduce the sensitivity/recall for weak
similarities, but can increase precision. On the other hand, shorter words
may increase sensitivity or recall, but may reduce precision. Computation
time generally increases with shorter words and decreases with longer
words, but it increases again for very long words. Memory requirements for
a part of the index increase with a factor of 4 each time word length
increases by one nucleotide, and this generally becomes significant for
long words (12 or more). The default value is 8.
Shuffling options:
Fasta entries in the input file are outputted in a pseudo-random order.
--output filename
Write the shuffled sequences to filename, in fasta format.
--randseed positive integer
When shuffling sequence order, use integer as seed. A given seed always
produces the same output order (useful for replicability). Set to 0 to use
a pseudo-random seed (default behavior).
--relabel string
Relabel sequences using the prefix string and a ticker (1, 2, 3, etc.) to
construct the new headers. Use --sizeout to conserve the abundance
annotations.
--relabel_keep
When relabelling, keep the old identifier in the header after a space.
--relabel_md5
Relabel sequences using the MD5 message digest algorithm applied to each
sequence. Former sequence headers are discarded. The sequence is converted
to upper case and U is replaced by T before the digest is computed. The
MD5 digest is a cryptographic hash function designed to minimize the
probability that two different inputs gives the same output, even for very
similar, but non-identical inputs. Still, there is always a very small,
but non-zero probability that two different inputs give the same result.
The MD5 digest generates a 128-bit (16-byte) digest that is represented by
16 hexadecimal numbers (using 32 symbols among 0123456789abcdef). Use
--sizeout to conserve the abundance annotations.
--relabel_self
Relabel sequences using the sequence itself as the label.
--relabel_sha1
Relabel sequences using the SHA1 message digest algorithm applied to each
sequence. It is similar to the --relabel_md5 option but uses the SHA1
algorithm instead of the MD5 algorithm. The SHA1 digest generates a
160-bit (20-byte) result that is represented by 20 hexadecimal numbers (40
symbols). The probability of a collision (two non-identical sequences
having the same digest) is smaller for the SHA1 algorithm than it is for
the MD5 algorithm. Use --sizeout to conserve the abundance annotations.
--sizeout
When using --relabel, --relabel_self, --relabel_md5 or --relabel_sha1,
preserve and report abundance annotations to the output fasta file (using
the pattern ';size=integer;').
--shuffle filename
Pseudo-randomly shuffle the order of sequences contained in filename.
--topn positive integer
Output only the first integer sequences after pseudo-random reordering.
--xsize Strip abundance information from the headers when writing the output file.
Sorting options:
Fasta entries are sorted by decreasing abundance (--sortbysize) or sequence length
(--sortbylength). To obtain a stable sorting order, ties are sorted by decreasing
abundance and label increasing alpha-numerical order (--sortbylength), or just by
label increasing alpha-numerical order (--sortbysize). Label sorting assumes that
all sequences have unique labels. The same applies to the automatic sorting
performed during chimera checking (--uchime_denovo), dereplication
(--derep_fulllength), and clustering (--cluster_fast and --cluster_size).
--maxsize positive integer
When using --sortbysize, discard sequences with an abundance value greater
than integer.
--minsize positive integer
When using --sortbysize, discard sequences with an abundance value smaller
than integer.
--output filename
Write the sorted sequences to filename, in fasta format.
--relabel string
Please see the description of the same option under Chimera detection for
details.
--relabel_keep
When relabelling, keep the old identifier in the header after a space.
--relabel_md5
Please see the description of the same option under Chimera detection for
details.
--relabel_self
Please see the description of the same option under Chimera detection for
details.
--relabel_sha1
Please see the description of the same option under Chimera detection for
details.
--sizeout
When using --relabel, report abundance annotations to the output fasta
file (using the pattern ';size=integer;').
--sortbylength filename
Sort by decreasing length the sequences contained in filename. See the
general options --minseqlength and --maxseqlength to eliminate short and
long sequences.
--sortbysize filename
Sort by decreasing abundance the sequences contained in filename (missing
abundance values are assumed to be ';size=1'). See the options --minsize
and --maxsize to eliminate rare and dominant sequences.
--topn positive integer
Output only the top integer sequences (i.e. the longest or the most
abundant).
--xsize Strip abundance information from the headers when writing the output file.
Subsampling options:
Subsampling randomly extracts a certain number or a certain percentage of the
sequences in the input file. If the --sizein option is in effect, the abundances of
the input sequences is taken into account and the sampling is performed as if the
input sequences were rereplicated, subsampled and dereplicated before being written
to the output file. The extraction is performed as a random sampling with a uniform
distribution among the input sequences and is performed without replacement. The
input file is specified with the --fastx_subsample option, the output files are
specified with the --fastaout and --fastqout options and the amount of sequences to
be sampled is specified with the --sample_pct or --sample_size options. The
sequences not sampled may be written to files specified with the options
--fasta_discarded and --fastq_discarded. The --fastq_ascii, --fastq_qmin and
--fastq_qmax options are also available.
--fastaout filename
Write the sampled sequences to filename, in fasta format.
--fastaout_discarded filename
Write the sequences not sampled to filename, in fasta format.
--fastq_ascii positive integer
Define the ASCII character number used as the basis for the FASTQ quality
score. The default is 33, which is used by the Sanger / Illumina 1.8+
FASTQ format (phred+33). The value 64 is used by the Solexa, Illumina 1.3+
and Illumina 1.5+ formats (phred+64). Only 33 and 64 are valid arguments.
--fastq_qmax positive integer
Specify the maximum quality score accepted when reading FASTQ files. The
default is 41, which is usual for recent Sanger/Illumina 1.8+ files.
--fastq_qmin positive integer
Specify the minimum quality score accepted for FASTQ files. The default is
0, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may
use scores between -5 and 2.
--fastqout filename
Write the sampled sequences to filename, in fastq format. Requires input
in fastq format.
--fastqout_discarded filename
Write the sequences not sampled to filename, in fastq format. Requires
input in fastq format.
--fastx_subsample filename
Perform subsampling from the sequences in the specified input file that is
in FASTA or FASTQ format.
--randseed positive integer
Use integer as a seed for the pseudo-random generator. A given seed always
produces the same output, which is useful for replicability. Set to 0 to
use a pseudo-random seed (default behavior).
--relabel string
Relabel sequences using the prefix string and a ticker (1, 2, 3, etc.) to
construct the new headers. Use --sizeout to conserve the abundance
annotations.
--relabel_keep
When relabelling, keep the old identifier in the header after a space.
--relabel_md5
Relabel sequences using the MD5 message digest algorithm applied to each
sequence. Former sequence headers are discarded. The sequence is converted
to upper case and U is replaced by T before the digest is computed. The
MD5 digest is a cryptographic hash function designed to minimize the
probability that two different inputs give the same output, even for very
similar, but non-identical inputs. Still, there is always a very small,
but non-zero probability that two different inputs give the same result.
The MD5 digest generates a 128-bit (16-byte) digest that is represented by
16 hexadecimal numbers (using 32 symbols among 0123456789abcdef). Use
--sizeout to conserve the abundance annotations.
--relabel_self
Relabel sequences using the sequence itself as the label.
--relabel_sha1
Relabel sequences using the SHA1 message digest algorithm applied to each
sequence. It is similar to the --relabel_md5 option but uses the SHA1
algorithm instead of the MD5 algorithm. The SHA1 digest generates a
160-bit (20-byte) result that is represented by 20 hexadecimal numbers (40
symbols). The probability of a collision (two non-identical sequences
having the same digest) is smaller for the SHA1 algorithm than it is for
the MD5 algorithm. Use --sizeout to conserve the abundance annotations.
--sample_pct real
Subsample the given percentage of the input sequences. Accepted values
range from 0.0 to 100.0.
--sample_size positive integer
Extract the given number of sequences.
--sizein Take the abundance information of the input file into account, otherwise
the abundance of each sequence is considered to be 1.
--sizeout
Write abundance information to the output file.
--xsize Strip abundance information from the headers when writing the output file.
Taxonomic classification options:
The vsearch command --sintax will classify the input sequences according to the
Sintax algorithm as described by Robert Edgar (2016) in SINTAX: a simple non-
Bayesian taxonomy classifier for 16S and ITS sequences, BioRxiv, 074161. Preprint.
doi: 10.1101/074161
The name of the fasta file containing the input sequences to be classified is given
as an argument to the --sintax command. The reference sequence database is
specified with the --db option. The results are written in a tab delimited text
file whose name is specified with the --tabbedout option. The --sintax_cutoff
option may be used to set a minimum level of bootstrap support for the taxonomic
ranks to be reported. The `--randseed` option may be included to specify a seed for
initialisation of the random number generator used by the algorithm.
Multithreading is supported. Databases in UDB files are supported. The strand
option may be specified.
The reference database must contain taxonomic information in the header of each
sequence in the form of a string starting with ";tax=" and followed by a comma-
separated list of up to eight taxonomic identifiers. Each taxonomic identifier must
start with an indication of the rank by one of the letters d (for domain) k
(kingdom), p (phylum), c (class), o (order), f (family), g (genus), or s (species).
The letter is followed by a colon (:) and the name of that rank. Commas and
semicolons are not allowed in the name of the rank.
Example: ">X80725_S000004313;tax=d:Bacteria,p:Proteobacteria,c:Gammaproteobacteria,
o:Enterobacteriales,f:Enterobacteriaceae,g:Escherichia/Shigella,
s:Escherichia_coli".
The option --notrunclabels is turned on by default for this command, allowing
spaces in the taxonomic identifiers.
--db filename
Read the reference sequences from filename, in FASTA, FASTQ or UDB format.
These sequences need to be annotated with taxonomy.
--randseed positive integer
Use integer as seed for the random number generator used in the Sintax
algorithm. A given seed always produces the same output order (useful for
replicability). Set to 0 to use a pseudo-random seed (default behavior).
--sintax_cutoff real
Specify a minimum level of bootstrap support for the taxonomic ranks that
will be included in column 4 of the output file. For instance 0.9,
corresponding to 90%.
--sintax filename
Read the input sequences from filename, in FASTA or FASTQ format.
--tabbedout filename
Write the results to filename, in a tab-separated text format. Column 1
contains the query label. Column 2 contains the predicted taxonomy in the
same format as for the reference data, with bootstrap support indicated in
parentheses after each rank. Column 3 contains the strand. If the
--sintax_cutoff option is used, the predicted taxonomy will be repeated in
column 4 while omitting the bootstrap values and including only the ranks
with support at or above the threshold.
UDB options:
Databases to be used with the --usearch_global command may be prepared from FASTA
files and stored to a binary UDB formatted file in order to speed up searching.
This may be worthwhile when searching a large database repeatedly. The sequences
are indexed and stored in a way that can be quickly loaded into memory. The
commands and options below can be used to create and inspect UDB files. An UDB file
may be specified with the --db option instead of a FASTA formatted file with the
--usearch_global command.
--dbmask none|dust|soft
Specify the sequence masking method used with the --makeudb_usearch
command, either none, dust or soft. No masking is performed when none is
specified. When dust is specified, the DUST algorithm will be used for
masking low complexity regions (short repeats and skewed composition).
Lower case letters in the input file will be masked when soft is specified
(soft masking).
--hardmask
Mask sequences by replacing letters with N for the --makeudb_usearch
command. The default is to use lower case letters (soft masking).
--makeudb_usearch filename
Create an UDB database file from the FASTA-formatted sequences in the file
with the given filename. The UDB database is written to the file specified
with the --output option.
--output filename
Specify the filename of a FASTA or UDB output file for the
--makeudb_usearch or the --udb2fasta command, respectively.
--udb2fasta filename
Read the UDB database in the file with the given filename and output the
sequences in FASTA format in the file specified by the --output option.
--udbinfo filename
Show information about the UDB database in the file with the given
filename.
--udbstats filename
Report statistics about the indexed words in the UDB database in the file
with the given filename.
--wordlength positive integer
Specify the length of the words to be used when creating the UDB database
index using the --makeudb_usearch command. Valid numbers range from 3 to
15. The default is 8.
Userfields (fields accepted by the --userfields option):
aln Print a string of M (match/mismatch, i.e. not a gap), D (delete, i.e. a
gap in the query) and I (insert, i.e. a gap in the target) representing
the pairwise alignment. Empty field if there is no alignment.
alnlen Print the length of the query-target alignment (number of columns). The
field is set to 0 if there is no alignment.
bits Bit score (not computed for nucleotide alignments). Always set to 0.
caln Compact representation of the pairwise alignment using the CIGAR format
(Compact Idiosyncratic Gapped Alignment Report): M (match/mismatch), D
(deletion) and I (insertion). Empty field if there is no alignment.
evalue E-value (not computed for nucleotide alignments). Always set to -1.
exts Number of columns containing a gap extension (zero or positive integer
value).
gaps Number of columns containing a gap (zero or positive integer value).
id The percentage of identity, according to the identity definition specified
by the --iddef option. Equal to id0, id1, id2, id3 or id4 below. By
default the same as id2.
id0 CD-HIT definition of the percentage of identity (real value ranging from
0.0 to 100.0) using the length of the shortest sequence in the pairwise
alignment as denominator: 100 * (matching columns) / (shortest sequence
length).
id1 The percentage of identity (real value ranging from 0.0 to 100.0) is
defined as the edit distance: 100 * (matching columns) / (alignment
length).
id2 The percentage of identity (real value ranging from 0.0 to 100.0) is
defined as the edit distance, excluding terminal gaps.
id3 Marine Biological Lab definition of the percentage of identity (real value
ranging from 0.0 to 100.0), counting each gap opening (internal or
terminal) as a single mismatch, whether or not the gap was extended, and
using the length of the longest sequence in the pairwise alignment as
denominator: 100 * (1.0 - [(mismatches + gaps) / (longest sequence
length)]).
id4 BLAST definition of the percentage of identity (real value ranging from
0.0 to 100.0), equivalent to --iddef 1 in a context of global pairwise
alignment. The field id4 is always equal to the field id1.
ids Number of matches in the alignment (zero or positive integer value).
mism Number of mismatches in the alignment (zero or positive integer value).
opens Number of columns containing a gap opening (zero or positive integer
value).
pairs Number of columns containing only nucleotides. That value corresponds to
the length of the alignment minus the gap-containing columns (zero or
positive integer value).
pctgaps Number of columns containing gaps expressed as a percentage of the
alignment length (real value ranging from 0.0 to 100.0).
pctpv Percentage of positive columns. When working with nucleotide sequences,
this is equivalent to the percentage of matches (real value ranging from
0.0 to 100.0).
pv Number of positive columns. When working with nucleotide sequences, this
is equivalent to the number of matches (zero or positive integer value).
qcov Fraction of the query sequence that is aligned with the target sequence
(real value ranging from 0.0 to 100.0). The query coverage is computed as
100.0 * (matches + mismatches) / query sequence length. Internal or
terminal gaps are not taken into account. The field is set to 0.0 if there
is no alignment.
qframe Query frame (-3 to +3). That field only concerns coding sequences and is
not computed by vsearch. Always set to +0.
qhi Last nucleotide of the query aligned with the target. Always equal to the
length of the pairwise alignment, 0 otherwise (see qihi to ignore terminal
gaps).
qihi Last nucleotide of the query aligned with the target (ignoring terminal
gaps). Nucleotide numbering starts from 1. The field is set to 0 if there
is no alignment.
qilo First nucleotide of the query aligned with the target (ignoring initial
gaps). Nucleotide numbering starts from 1. The field is set to 0 if there
is no alignment.
ql Query sequence length (positive integer value). The field is set to 0 if
there is no alignment.
qlo First nucleotide of the query aligned with the target. Always equal to 1
if there is an alignment, 0 otherwise (see qilo to ignore initial gaps).
qrow Print the sequence of the query segment as seen in the pairwise alignment
(i.e. with gap insertions if need be). Empty field if there is no
alignment.
qs Query segment length. Always equal to query sequence length.
qstrand Query strand orientation (+ or - for nucleotide sequences). Empty field if
there is no alignment.
query Query label.
raw Raw alignment score (negative, null or positive integer value). The score
is the sum of match rewards minus mismatch penalties, gap openings and gap
extensions. The field is set to 0 if there is no alignment.
target Target label. The field is set to '*' if there is no alignment.
tcov Fraction of the target sequence that is aligned with the query sequence
(real value ranging from 0.0 to 100.0). The target coverage is computed as
100.0 * (matches + mismatches) / target sequence length. Internal or
terminal gaps are not taken into account. The field is set to 0.0 if
there is no alignment.
tframe Target frame (-3 to +3). That field only concerns coding sequences and is
not computed by vsearch. Always set to +0.
thi Last nucleotide of the target aligned with the query. Always equal to the
length of the pairwise alignment, 0 otherwise (see tihi to ignore terminal
gaps).
tihi Last nucleotide of the target aligned with the query (ignoring terminal
gaps). Nucleotide numbering starts from 1. The field is set to 0 if there
is no alignment.
tilo First nucleotide of the target aligned with the query (ignoring initial
gaps). Nucleotide numbering starts from 1. The field is set to 0 if there
is no alignment.
tl Target sequence length (positive integer value). The field is set to 0 if
there is no alignment.
tlo First nucleotide of the target aligned with the query. Always equal to 1
if there is an alignment, 0 otherwise (see tilo to ignore initial gaps).
trow Print the sequence of the target segment as seen in the pairwise alignment
(i.e. with gap insertions if need be). Empty field if there is no
alignment.
ts Target segment length. Always equal to target sequence length. The field
is set to 0 if there is no alignment.
tstrand Target strand orientation (+ or - for nucleotide sequences). Always set to
'+', so reverse strand matches have tstrand '+' and qstrand '-'. Empty
field if there is no alignment.
DELIBERATE CHANGES
If you are a usearch user, our objective is to make you feel at home. That's why vsearch
was designed to behave like usearch, to some extent. Like any complex software, usearch is
not free from quirks and inconsistencies. We decided not to reproduce some of them, and
for complete transparency, to document here the deliberate changes we made.
During a search with usearch, when using the options --blast6out and --output_no_hits, for
queries with no match the number of fields reported is 13, where it should be 12. This is
corrected in vsearch.
The field raw of the --userfields option is not informative in usearch. This is corrected
in vsearch.
The fields qlo, qhi, tlo, thi now have counterparts (qilo, qihi, tilo, tihi) reporting
alignment coordinates ignoring terminal gaps.
In usearch, when using the option --output_no_hits, queries that receive no match are
reported in --blast6out file, but not in the alignment output file. This is corrected in
vsearch.
vsearch introduces a new --cluster_size command that sorts sequences by decreasing
abundance before clustering.
vsearch reintroduces --iddef alternative pairwise identity definitions that were removed
from usearch.
vsearch extends the --topn option to sorting commands.
vsearch extends the --sizein option to dereplication (--derep_fulllength) and clustering
(--cluster_fast).
vsearch treats T and U as identical nucleotides during dereplication.
vsearch sorting is stabilized by using sequence abundances or sequences labels as
secondary or tertiary keys.
vsearch by default uses the DUST algorithm for masking low-complexity regions. Masking
behavior is also slightly changed to be more consistent.
NOVELTIES
vsearch introduces new commands and new options not present in usearch 7. They are
described in the 'Options' section of this manual. Here is a short list:
- uchime2_denovo, uchime3_denovo, alignwidth, borderline, fasta_score (chimera
checking)
- cluster_size, cluster_unoise, clusterout_id, clusterout_sort, profile
(clustering)
- fasta_width, gzip_decompress, bzip2_decompress (general option)
- iddef (clustering, pairwise alignment, searching)
- maxuniquesize (dereplication)
- relabel_md5, relabel_self and relabel_sha1 (chimera detection, dereplication,
FASTQ processing, shuffling, sorting)
- shuffle (shuffling)
- fastq_eestats, fastq_eestats2, fastq_maxlen, fastq_truncee (FASTQ processing)
- fastaout_discarded, fastqout_discarded (subsampling)
- rereplicate (dereplication/rereplication)
EXAMPLES
Align all sequences in a database with each other and output all pairwise alignments:
vsearch --allpairs_global database.fas --alnout results.aln --acceptall
Check for the presence of chimeras (de novo); parents should be at least 1.5 times more
abundant than chimeras. Output non-chimeric sequences in fasta format (no wrapping):
vsearch --uchime_denovo queries.fas --abskew 1.5 --nonchimeras results.fas
--fasta_width 0
Cluster with a 97% similarity threshold, collect cluster centroids, and write cluster
descriptions using a uclust-like format:
vsearch --cluster_fast queries.fas --id 0.97 --centroids centroids.fas --uc
clusters.uc
Dereplicate the sequences contained in queries.fas, take into account the abundance
information already present, write unwrapped fasta sequences to queries_unique.fas with
the new abundance information, discard all sequences with an abundance of 1:
vsearch --derep_fulllength queries.fas --sizein --fasta_width 0 --sizeout --output
queries_unique.fas --minuniquesize 2
Mask simple repeats and low complexity regions in the input fasta file with the DUST
algorithm (masked regions are lowercased), and write the results to the output file:
vsearch --maskfasta queries.fas --qmask dust --output queries_masked.fas
Search queries in a reference database, with a 80%-similarity threshold, take terminal
gaps into account when calculating pairwise similarities, output pairwise alignments:
vsearch --usearch_global queries.fas --db references.fas --id 0.8 --iddef 1
--alnout results.aln
Search a sequence dataset against itself (ignore self hits), get all matches with at least
60% similarity, and collect results in a blast-like tab-separated format. Accept an
unlimited number of hits (--maxaccepts 0), and compare each query to all other sequences,
including unlikely candidates (--maxrejects 0):
vsearch --usearch_global queries.fas --db queries.fas --self --id 0.6 --blast6out
results.blast6 --maxaccepts 0 --maxrejects 0
Shuffle the input fasta file (change the order of sequences) in a repeatable fashion
(fixed seed), and write unwrapped fasta sequences to the output file:
vsearch --shuffle queries.fas --output queries_shuffled.fas --randseed 13
--fasta_width 0
Sort by decreasing abundance the sequences contained in queries.fas (using the
'size=integer' information), relabel the sequences while preserving the abundance
information (with --sizeout), keep only sequences with an abundance equal to or greater
than 2:
vsearch --sortbysize queries.fas --output queries_sorted.fas --relabel sampleA_
--sizeout --minsize 2
AUTHORS
Implementation by Torbjørn Rognes and Tomás Flouri, documentation by Frédéric Mahé.
CITATION
Rognes T, Flouri T, Nichols B, Quince C, Mahé F. (2016) VSEARCH: a versatile open source
tool for metagenomics. PeerJ 4:e2584 doi: 10.7717/peerj.2584
REPORTING BUGS
Submit suggestions and bug-reports at <https://github.com/torognes/vsearch/issues>, send a
pull request on <https://github.com/torognes/vsearch>, or compose a friendly or
curmudgeont e-mail to Torbjørn Rognes <torognes@ifi.uio.no>.
AVAILABILITY
Source code and binaries are available at <https://github.com/torognes/vsearch>.
COPYRIGHT
Copyright (C) 2014-2021, Torbjørn Rognes, Frédéric Mahé and Tomás Flouri
All rights reserved.
Contact: Torbjørn Rognes <torognes@ifi.uio.no>, Department of Informatics, University of
Oslo, PO Box 1080 Blindern, NO-0316 Oslo, Norway
This software is dual-licensed and available under a choice of one of two licenses, either
under the terms of the GNU General Public License version 3 or the BSD 2-Clause License.
GNU General Public License version 3
This program is free software: you can redistribute it and/or modify it under the terms of
the GNU General Public License as published by the Free Software Foundation, either
version 3 of the License, or (at your option) any later version.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY;
without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.
See the GNU General Public License for more details.
You should have received a copy of the GNU General Public License along with this program.
If not, see <http://www.gnu.org/licenses/>.
The BSD 2-Clause License
Redistribution and use in source and binary forms, with or without modification, are
permitted provided that the following conditions are met:
1. Redistributions of source code must retain the above copyright notice, this list of
conditions and the following disclaimer.
2. Redistributions in binary form must reproduce the above copyright notice, this list of
conditions and the following disclaimer in the documentation and/or other materials
provided with the distribution.
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND ANY
EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE
COPYRIGHT HOLDER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL,
EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF
SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION)
HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR
TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS
SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
We would like to thank the authors of the following projects for making their source code
available:
- vsearch includes code from Google's CityHash project by Geoff Pike and Jyrki
Alakuijala, providing some excellent hash functions available under a MIT
license.
- vsearch includes code derived from Tatusov and Lipman's DUST program that is in
the public domain.
- vsearch includes public domain code written by Alexander Peslyak for the MD5
message digest algorithm.
- vsearch includes public domain code written by Steve Reid and others for the SHA1
message digest algorithm.
- vsearch binaries may include code from the zlib library, copyright Jean-Loup
Gailly and Mark Adler.
- vsearch binaries may include code from the bzip2 library, copyright Julian R.
Seward.
SEE ALSO
swipe, an extremely fast pairwise local (Smith-Waterman) database search tool by Torbjørn
Rognes, available at <https://github.com/torognes/swipe>.
swarm, a fast and accurate amplicon clustering method by Frédéric Mahé and Torbjørn
Rognes, available at <https://github.com/torognes/swarm>.
VERSION HISTORY
New features and important modifications of vsearch (short lived or minor bug releases may
not be mentioned):
v1.0.0 released November 28th, 2014
First public release.
v1.0.1 released December 1st, 2014
Bug fixes (sortbysize, semicolon after size annotation in headers) and minor
changes (labels as secondary sort key for most sorts, treat T and U as identical
for dereplication, only output size in --dbmatched file if --sizeout specified).
v1.0.2 released December 6th, 2014
Bug fixes (ssse3/sse4.1 requirement, memory leak).
v1.0.3 released December 6th, 2014
Bug fix (now writes help to stdout instead of stderr).
v1.0.4 released December 8th, 2014
Added --allpairs_global option. Reduce memory requirements slightly and eliminate
memory leaks.
v1.0.5 released December 9th, 2014
Fixes a minor bug with --allpairs_global and --acceptall options.
v1.0.6 released December 14th, 2014
Fixes a memory allocation bug in chimera detection (--uchime_ref option).
v1.0.7 released December 19th, 2014
Fixes a bug in the output from chimera detection with the --uchimeout option.
v1.0.8 released January 22nd, 2015
Introduces several changes and bug fixes:
- a new linear memory aligner for alignment of sequences longer than 5,000
nucleotides,
- a new --cluster_size command that sorts sequences by decreasing abundance before
clustering,
- meaning of userfields qlo, qhi, tlo, thi changed for compatibility with usearch,
- new userfields qilo, qihi, tilo, tihi give alignment coordinates ignoring
terminal gaps,
- in --uc output files, a perfect alignment is indicated with a '=' sign,
- the option --cluster_fast now sorts sequences by decreasing length, then by
decreasing abundance and finally by sequence identifier,
- default --maxseqlength value set to 50,000 nucleotides,
- fix for bug in alignment in rare cases,
- fix for lack of detection of under- or overflow in SIMD aligner.
v1.0.9 released January 22nd, 2015
Fixes a bug in the function sorting sequences by decreasing abundance
(--sortbysize).
v1.0.10 released January 23rd, 2015
Fixes a bug where the --sizein option was ignored and always treated as on,
affecting clustering and dereplication commands.
v1.0.11 released February 5th, 2015
Introduces the possibility to output results in SAM format (for clustering,
pairwise alignment and searching).
v1.0.12 released February 6th, 2015
Temporarily fixes a problem with long headers in FASTA files.
v1.0.13 released February 17th, 2015
Fix a memory allocation problem when computing multiple sequence alignments with
the --msaout and --consout options, as well as a memory leak. Also increased line
buffer for reading FASTA files to 4MB.
v1.0.14 released February 17th, 2015
Fix a bug where the multiple alignment and consensus sequence computed after
clustering ignored the strand of the sequences. Also decreased size of line buffer
for reading FASTA files to 1MB again due to excessive stack memory usage.
v1.0.15 released February 18th, 2015
Fix bug in calculation of identity metric between sequences when using the MBL
definition (--iddef 3).
v1.0.16 released February 19th, 2015
Integrated patches from Debian for increased compatibility with various
architectures.
v1.1.0 released February 20th, 2015
Added the --quiet option to suppress all output to stdout and stderr except for
warnings and fatal errors. Added the --log option to write messages to a log file.
v1.1.1 released February 20th, 2015
Added info about --log and --quiet options to help text.
v1.1.2 released March 18th, 2015
Fix bug with large datasets. Fix format of help info.
v1.1.3 released March 18th, 2015
Fix more bugs with large datasets.
v1.2.0-1.2.19 released July 6th to September 8th, 2015
Several new commands and options added. Bugs fixed. Documentation updated.
v1.3.0 released September 9th, 2015
Changed to autotools build system.
v1.3.1 released September 14th, 2015
Several new commands and options. Bug fixes.
v1.3.2 released September 15th, 2015
Fixed memory leaks. Added '-h' shortcut for help. Removed extra 'v' in version
number.
v1.3.3 released September 15th, 2015
Fixed bug in hexadecimal digits of MD5 and SHA1 digests. Added --samheader option.
v1.3.4 released September 16th, 2015
Fixed compilation problems with zlib and bzip2lib.
v1.3.5 released September 17th, 2015
Minor configuration/makefile changes to compile to native CPU and simplify
makefile.
v1.4.0 released September 25th, 2015
Added --sizeorder option.
v1.4.1 released September 29th, 2015
Inserted public domain MD5 and SHA1 code to eliminate dependency on crypto and
openssl libraries and their licensing issues.
v1.4.2 released October 2nd, 2015
Dynamic loading of libraries for reading gzip and bzip2 compressed files if
available. Circumvention of missing gzoffset function in zlib 1.2.3 and earlier.
v1.4.3 released October 3rd, 2015
Fix a bug with determining amount of memory on some versions of Apple OS X.
v1.4.4 released October 3rd, 2015
Remove debug message.
v1.4.5 released October 6th, 2015
Fix memory allocation bug when reading long FASTA sequences.
v1.4.6 released October 6th, 2015
Fix subtle bug in SIMD alignment code that reduced accuracy.
v1.4.7 released October 7th, 2015
Fixes a problem with searching for or clustering sequences with repeats. In this
new version, vsearch looks at all words occurring at least once in the sequences in
the initial step. Previously only words occurring exactly once were considered. In
addition, vsearch now requires at least 10 words to be shared by the sequences,
previously only 6 were required. If the query contains less than 10 words, all
words must be present for a match. This change seems to lead to slightly reduced
recall, but somewhat increased precision, ending up with slightly improved overall
accuracy.
v1.5.0 released October 7th, 2015
This version introduces the new option --minwordmatches that allows the user to
specify the minimum number of matching unique words before a sequence is considered
further. New default values for different word lengths are also set. The minimum
word length is increased to 7.
v1.6.0 released October 9th, 2015
This version adds the relabeling options (--relabel, --relabel_md5 and
--relabel_sha1) to the shuffle command. It also adds the --xsize option to the
clustering, dereplication, shuffling and sorting commands.
v1.6.1 released October 14th, 2015
Fix bugs and update manual and help text regarding relabelling. Add all relabelling
options to the subsampling command. Add the --xsize option to chimera detection,
dereplication and fastq filtering commands. Refactoring of code.
v1.7.0 released October 14th, 2015
Add --relabel_keep option.
v1.8.0 released October 19th, 2015
Added --search_exact, --fastx_mask and --fastq_convert commands. Changed most
commands to read FASTQ input files as well as FASTA files. Modified
--fastx_revcomp and --fastx_subsample to write FASTQ files.
v1.8.1 released November 2nd, 2015
Fixes for compatibility with QIIME and older OS X versions.
v1.9.0 released November 12th, 2015
Added the --fastq_mergepairs command and associated options. This command has not
been tested well yet. Included additional files to avoid dependency of autoconf for
compilation. Fixed an error where identifiers in fasta headers where not truncated
at tabs, just spaces. Fixed a bug in detection of the file format (FASTA/FASTQ) of
a gzip compressed input file.
v1.9.1 released November 13th, 2015
Fixed memory leak and a bug in score computation in --fastq_mergepairs, and
improved speed.
v1.9.2 released November 17th, 2015
Fixed a bug in the computation of some values with --fastq_stats.
v1.9.3 released November 19th, 2015
Workaround for missing x86intrin.h with old compilers.
v1.9.4 released December 3rd, 2015
Fixed incrementation of counter when relabeling dereplicated sequences.
v1.9.5 released December 3rd, 2015
Fixed bug resulting in inferior chimera detection performance.
v1.9.6 released January 8th, 2016
Fixed bug in aligned sequences produced with --fastapairs and --userout (qrow,
trow) options.
v1.9.7 released January 12th, 2016
Masking behavior is changed somewhat to keep the letter case of the input sequences
unchanged when no masking is performed. Masking is now performed also during
chimera detection. Documentation updated.
v1.9.8 released January 22nd, 2016
Fixed bug causing segfault when chimera detection is performed on extremely short
sequences.
v1.9.9 released January 22nd, 2016
Adjusted default minimum number of word matches during searches for improved
performance.
v1.9.10 released January 25th, 2016
Fixed bug related to masking and lower case database sequences.
v1.10.0 released February 11th, 2016
Parallelized and improved merging of paired-end reads and adjusted some defaults.
Removed progress indicator when stderr is not a terminal. Added --fasta_score
option to report chimera scores in FASTA files. Added --rereplicate and
--fastq_eestats commands. Fixed typos. Added relabelling to files produced with
--consout and --profile options.
v1.10.1 released February 23rd, 2016
Fixed a bug affecting the --fastq_mergepairs command causing FASTQ headers to be
truncated at first space (despite the bug fix release 1.9.0 of November 12th,
2015). Full headers are now included in the output (no matter if --notrunclabels is
in effect or not).
v1.10.2 released March 18th, 2016
Fixed a bug causing a segmentation fault when running --usearch_global with an
empty query sequence. Also fixed a bug causing imperfect alignments to be reported
with an alignment string of '=' in uc output files. Fixed typos in man file. Fixed
fasta/fastq processing code regarding presence or absence of compression library
header files.
v1.11.1 released April 13th, 2016
Added strand information in UC file for --derep_fulllength and --derep_prefix.
Added expected errors (ee) to header of FASTA files specified with --fastaout and
--fastaout_discarded when --eeout or --fastq_eeout option is in effect for
fastq_filter and fastq_mergepairs. The options --eeout and --fastq_eeout are now
equivalent.
v1.11.2 released June 21st, 2016
Two bugs were fixed. The first issue was related to the --query_cov option that
used a different coverage definition than the qcov userfield. The coverage is now
defined as the fraction of the whole query sequence length that is aligned with
matching or mismatching residues in the target. All gaps are ignored. The other
issue was related to the consensus sequences produced during clustering when only
N's were present in some positions. Previously these would be converted to A's in
the consensus. The behaviour is changed so that N's are produced in the consensus,
and it should now be more compatible with usearch.
v2.0.0 released June 24th, 2016
This major new version supports reading from pipes. Two new options are added:
--gzip_decompress and --bzip2_decompress. One of these options must be specified if
reading compressed input from a pipe, but are not required when reading from
ordinary files. The vsearch header that was previously written to stdout is now
written to stderr. This enables piping of results for further processing. The file
name '-' now represent standard input (/dev/stdin) or standard output (/dev/stdout)
when reading or writing files, respectively. Code for reading FASTA and FASTQ files
has been refactored.
v2.0.1 released June 30th, 2016
Avoid segmentation fault when masking very long sequences.
v2.0.2 released July 5th, 2016
Avoid warnings when compiling with GCC 6.
v2.0.3 released August 2nd, 2016
Fixed bad compiler options resulting in Illegal instruction errors when running
precompiled binaries.
v2.0.4 released September 1st, 2016
Improved error message for bad FASTQ quality values. Improved manual.
v2.0.5 released September 9th, 2016
Add options --fastaout_discarded and --fastqout_discarded to output discarded
sequences from subsampling to separate files. Updated manual.
v2.1.0 released September 16th, 2016
New command: --fastx_filter. New options: --fastq_maxlen, --fastq_truncee. Allow
--minwordmatches down to 3.
v2.1.1 released September 23rd, 2016
Fixed bugs in output to UC-files. Improved help text and manual.
v2.1.2 released September 28th, 2016
Fixed incorrect abundance output from fastx_filter and fastq_filter when
relabelling.
v2.2.0 released October 7th, 2016
Added OTU table generation options --biomout, --mothur_shared_out and --otutabout
to the clustering and searching commands.
v2.3.0 released October 10th, 2016
Allowed zero-length sequences in FASTA and FASTQ files. Added --fastq_trunclen_keep
option. Fixed bug with output of OTU tables to pipes.
v2.3.1 released November 16th, 2016
Fixed bug where --minwordmatches 0 was interpreted as the default minimum word
matches for the given word length instead of zero. When used in combination with
--maxaccepts 0 and --maxrejects 0 it will allow complete bypass of kmer-based
heuristics.
v2.3.2 released November 18th, 2016
Fixed bug where vsearch reported the ordinal number of the target sequence instead
of the cluster number in column 2 on H-lines in the uc output file after
clustering. For search and alignment commands both usearch and vsearch reports the
target sequence number here.
v2.3.3 released December 5th, 2016
A minor speed improvement.
v2.3.4 released December 9th, 2016
Fixed bug in output of sequence profiles and updated documentation.
v2.4.0 released February 8th, 2017
Added support for Linux on Power8 systems (ppc64le) and Windows on x86_64. Improved
detection of pipes when reading FASTA and FASTQ files. Corrected option for
specifying output from fastq_eestats command in help text.
v2.4.1 released March 1st, 2017
Fixed an overflow bug in fastq_stats and fastq_eestats affecting analysis of very
large FASTQ files. Fixed maximum memory usage reporting on Windows.
v2.4.2 released March 10th, 2017
Default value for fastq_minovlen increased to 16 in accordance with help text and
for compatibility with usearch. Minor changes for improved accuracy of paired-end
read merging.
v2.4.3 released April 6th, 2017
Fixed bug with progress bar for shuffling. Fixed missing N-lines in UC files with
usearch_global, search_exact and allpairs_global when the output_no_hits option was
not specified.
v2.4.4 released August 28th, 2017
Fixed a few minor bugs, improved error messages and updated documentation.
v2.5.0 released October 5th, 2017
Support for UDB database files. New commands: fastq_stripright, fastq_eestats2,
makeudb_usearch, udb2fasta, udbinfo, and udbstats. New general option: no_progress.
New options minsize and maxsize to fastx_filter. Minor bug fixes, error message
improvements and documentation updates.
v2.5.1 released October 25th, 2017
Fixed bug with bad default value of 1 instead of 32 for minseqlength when using the
makeudb_usearch command.
v2.5.2 released October 30th, 2017
Fixed bug with where '-' as an argument to the fastq_eestats2 option was treated
literally instead of equivalent to stdin.
v2.6.0 released November 10th, 2017
Rewritten paired-end reads merger with improved accuracy. Decreased default value
for fastq_minovlen option from 16 to 10. The default value for the fastq_maxdiffs
option is increased from 5 to 10. There are now other more important restrictions
that will avoid merging reads that cannot be reliably aligned.
v2.6.1 released December 8th, 2017
Improved parallelisation of paired end reads merging.
v2.6.2 released December 18th, 2017
Fixed option xsize that was partially inactive for commands uchime_denovo,
uchime_ref, and fastx_filter.
v2.7.0 released February 13th, 2018
Added commands cluster_unoise, uchime2_denovo and uchime3_denovo contributed by
Davide Albanese based on Robert Edgar's papers. Refactored fasta and fastq print
functions as well as code for extraction of abundance and other attributes from the
headers.
v2.7.1 released February 16th, 2018
Fix several bugs on Windows related to large files, use of "-" as a file name to
mean stdin or stdout, alignment errors, missed kmers and corrupted UDB files. Added
documentation of UDB-related commands.
v2.7.2 released April 20th, 2018
Added the sintax command for taxonomic classification. Fixed a bug with incorrect
FASTA headers of consensus sequences after clustering.
v2.8.0 released April 24th, 2018
Added the fastq_maxdiffpct option to the fastq_mergepairs command.
v2.8.1 released June 22nd, 2018
Fixes for compilation warnings with GCC 8.
v2.8.2 released August 21st, 2018
Fix for wrong placement of semicolons in header lines in some cases when using the
sizeout or xsize options. Reduced memory requirements for full-length dereplication
in cases with many duplicate sequences. Improved wording of fastq_mergepairs
report. Updated manual regarding use of sizein and sizeout with dereplication.
Changed a compiler option.
v2.8.3 released August 31st, 2018
Fix for segmentation fault for --derep_fulllength with --uc.
v2.8.4 released September 3rd, 2018
Further reduce memory requirements for dereplication when not using the uc option.
Fix output during subsampling when quiet or log options are in effect.
v2.8.5 released September 26th, 2018
Fixed a bug in fastq_eestats2 that caused the values for large lengths to be much
too high when the input sequences had varying lengths.
v2.8.6 released October 9th, 2018
Fixed a bug introduced in version 2.8.2 that caused derep_fulllength to include the
full FASTA header in its output instead of stopping at the first space (unless the
notrunclabels option is in effect).
v2.9.0 released October 10th, 2018
Added the fastq_join command.
v2.9.1 released October 29th, 2018
Changed compiler options that select the target cpu and tuning to allow the
software to run on any 64-bit x86 system, while tuning for more modern variants.
Avoid illegal instruction error on some architectures. Update documentation of
rereplicate command.
v2.10.0 released December 6th, 2018
Added the sff_convert command to convert SFF files to FASTQ. Added some additional
option argument checks. Fixed segmentation fault bug after some fatal errors when a
log file was specified.
v2.10.1 released December 7th, 2018
Improved sff_convert command. It will now read several variants of the SFF format.
It is also able to read from a pipe. Warnings are given if there are minor
problems. Errors messages have been improved. Minor speed and memory usage
improvements.
v2.10.2 released December 10th, 2018
Fixed bug in sintax with reversed order of domain and kingdom.
v2.10.3 released December 19th, 2018
Ported to Linux on ARMv8 (aarch64). Fixed compilation warning with gcc version
8.1.0 and 8.2.0.
v2.10.4 released January 4th, 2019
Fixed serious bug in x86_64 SIMD alignment code introduced in version 2.10.3. Added
link to BioConda in README. Fixed bug in fastq_stats with sequence length 1. Fixed
use of equals symbol in UC files for identical sequences with cluster_fast.
v2.11.0 released February 13th, 2019
Added ability to trim and filter paired-end reads using the reverse option with the
fastx_filter and fastq_filter commands. Added --xee option to remove ee attributes
from FASTA headers. Minor invisible improvement to the progress indicator.
v2.11.1 released February 28th, 2019
Minor change to the handling of the weak_id and id options when using
cluster_unoise.
v2.12.0 released March 19th, 2019
Take sequence abundance into account when computing consensus sequences or profiles
after clustering. Warn when rereplicating sequences without abundance info. Guess
offset 33 in more cases with fastq_chars. Stricter checking of option arguments and
option combinations.
v2.13.0 released April 11th, 2019
Added the --fastx_getseq, --fastx_getseqs and --fastx_getsubseq commands to extract
sequences from a FASTA or FASTQ file based on their labels. Improved handling of
ambiguous nucleotide symbols. Corrected behaviour of --uchime_ref command with and
options --self and --selfid. Strict detection of illegal options for each command.
v2.13.1 released April 26th, 2019
Minor changes to the allowed options for each command. All commands now allow the
log, quiet and threads options. If more than 1 thread is specified for commands
that are not multi-threaded, a warning will be issued. Minor changes to the manual.
v2.13.2 released April 30th, 2019
Fixed bug related to improper handling of newlines on Windows. Allowed option
strand plus to uchime_ref for compatibility.
v2.13.3 released April 30th, 2019
Fixed bug in FASTQ parsing introduced in version 2.13.2.
v2.13.4 released May 10th, 2019
Added information about support for gzip- and bzip2-compressed input files to the
output of the version command. Adapted source code for compilation on FreeBSD and
NetBSD systems.
v2.13.5 released July 2nd, 2019
Added cut command to fragment sequences at restriction sites. Silenced output from
the fastq_stats command if quiet option was given. Updated manual.
v2.13.6 released July 2nd, 2019
Added info about cut command to output of help command.
v2.13.7 released September 2nd, 2019
Fixed bug in consensus sequence introduced in version 2.13.0.
v2.14.0 released September 11th, 2019
Added relabel_self option. Made fasta_width, sizein, sizeout and relabelling
options valid for certain commands.
v2.14.1 released September 18th, 2019
Fixed bug with sequences written to file specified with fastaout_rev for commands
fastx_filter and fastq_filter.
v2.14.2 released January 28th, 2020
Fixed some issues with the cut, fastx_revcomp, fastq_convert, fastq_mergepairs, and
makeudb_usearch commands. Updated manual.
v2.15.0 released June 19th, 2020
Update manual and documentation. Turn on notrunclabels option for sintax command by
default. Change maxhits 0 to mean unlimited hits, like the default. Allow non-ascii
characters in headers, with a warning. Sort centroids and uc too when
clusterout_sort specified. Add cluster id to centroids output when clusterout_id
specified. Improve error messages when parsing FASTQ files. Add missing
fastq_qminout option and fix label_suffix option for fastq_mergepairs. Add derep_id
command that dereplicates based on both label and sequence. Remove compilation
warnings.
v2.15.1 released October 28th, 2020
Fix for dereplication when including reverse complement sequences and headers. Make
some extra checks when loading compression libraries and add more diagnostic output
about them to the output of the version command. Report an error when fastx_filter
is used with FASTA input and options that require FASTQ input. Update manual.
v2.15.2 released January 26th, 2021
No real functional changes, but some code and compilation changes. Compiles
successfully on macOS running on Apple Silicon (ARMv8). Binaries available. Code
updated for C++11. Minor adaptations for Windows compatibility, including the use
of the C++ standard library for regular expressions. Minor changes for
compatibility with Power8. Switch to C++ header files.
v2.16.0 released March 22nd, 2021
This version adds the orient command. It also handles empty input files properly.
Documentation has been updated.
v2.17.0 released March 29nd, 2021
The fastq_mergepairs command has been changed. It now allows merging of sequences
with overlaps as short as 5 bp if the --fastq_minovlen option has been adjusted
down from the default 10. In addition, much fewer pairs of reads should now be
rejected with the reason 'multiple potential alignments' as the algorithm for
detecting those have been changed.
v2.17.1 released June 14th, 2021
Modernized code. Minor changes to help info.
v2.18.0 released August 27th, 2021
Added the fasta2fastq command. Fixed search bug on ppc64le. Fixed bug with removal
of size and ee info in uc files. Fixed compilation errors in some cases. Made some
general code improvements. Updated manual.
v2.19.0 released December 21st, 2021
Added the lcaout and lca_cutoff options to enable the output of last common
ancestor (LCA) information about hits when searching. The randseed option was added
as a valid option to the sintax command. Code improvements.
v2.20.0 released January 10th, 2022
Added the fastx_uniques command and the fastq_qout_max option for dereplication of
FASTQ files. Some code cleaning.
v2.20.1 released January 11th, 2022
Fixes a bug in fastq_mergepair that caused an occational hang at the end when using
multiple threads.
v2.21.0 released January 12th, 2022
This version adds the sample, qsegout and tsegout options. It enables the use of
UDB databases with uchime_ref.
v2.21.1 released January 18th, 2022
Fix a problem with dereplication of empty input files. Update Altivec code on
ppc64le for improved compiler compatibility (vector->__vector).
v2.21.2 released September 12th, 2022
Fix problems with the lcaout option when using maxaccepts above 1 and either
lca_cutoff below 1 or with top_hits_only enabled. Update documentation. Update code
to avoid compiler warnings.
v2.22.0 released September 19th, 2022
Add the derep_smallmem command for dereplication using little memory.
v2.22.1 released September 19th, 2022
Fix compiler warning.