Provided by: trim-galore_0.6.10-1_all bug

NAME

       trim_galore - automate quality and adapter trimming for DNA sequencing

DESCRIPTION

              USAGE:

       trim_galore [options] <filename(s)>

       -h/--help               Print this help message and exits.

       -v/--version            Print the version information and exits.

       -q/--quality  <INT>       Trim low-quality ends from reads in addition to adapter removal.
       For

       RRBS samples, quality trimming will be performed first, and adapter
              trimming is carried in a second round. Other files are quality and adapter  trimmed
              in  a  single  pass. The algorithm is the same as the one used by BWA (Subtract INT
              from all qualities; compute partial sums  from  all  indices  to  the  end  of  the
              sequence;  cut  sequence  at  the index at which the sum is minimal). Default Phred
              score: 20.

       --phred33               Instructs Cutadapt to use ASCII+33 quality scores as Phred scores

              (Sanger/Illumina 1.9+ encoding) for quality trimming. Default: ON.

       --phred64               Instructs Cutadapt to use ASCII+64 quality scores as Phred scores

              (Illumina 1.5 encoding) for quality trimming.

       --fastqc                Run FastQC in the default mode on the FastQ file once trimming  is
       complete.

       --fastqc_args  "<ARGS>"  Passes extra arguments to FastQC. If more than one argument is to
       be passed

       to FastQC they must be in the form "arg1 arg2 etc.". An example would be:
              --fastqc_args  "--nogroup  --outdir   /home/".   Passing   extra   arguments   will
              automatically invoke FastQC, so --fastqc does not have to be specified separately.

       -a/--adapter  <STRING>   Adapter sequence to be trimmed. If not specified explicitly, Trim
       Galore will

       try to auto-detect whether the Illumina universal, Nextera transposase or Illumina
              small RNA adapter  sequence  was  used.  Also  see  '--illumina',  '--nextera'  and
              '--small_rna'.  If  no adapter can be detected within the first 1 million sequences
              of the first file specified or if there is a tie between several adapter sequences,
              Trim  Galore  defaults  to '--illumina' (as long as the Illumina adapter was one of
              the options, else '--nextera' is the default). A single base may also be  given  as
              e.g. -a A{10}, to be expanded to -a AAAAAAAAAA.

       -a2/--adapter2  <STRING>  Optional adapter sequence to be trimmed off read 2 of paired-end
       files. This

       option requires '--paired' to be specified as well. If the libraries to be trimmed
              are smallRNA then a2 will be set to the Illumina small RNA 5' adapter automatically
              (GATCGTCGGACT).  A  single base may also be given as e.g. -a2 A{10}, to be expanded
              to -a2 AAAAAAAAAA.

       --illumina              Adapter sequence to be trimmed is the first 13bp of  the  Illumina
       universal adapter

              'AGATCGGAAGAGC' instead of the default auto-detection of adapter sequence.

       --nextera                Adapter  sequence  to be trimmed is the first 12bp of the Nextera
       adapter

              'CTGTCTCTTATA' instead of the default auto-detection of adapter sequence.

       --small_rna             Adapter sequence to be trimmed is the first 12bp of  the  Illumina
       Small RNA 3' Adapter

       'TGGAATTCTCGG' instead of the default auto-detection of adapter sequence. Selecting
              to  trim  smallRNA  adapters  will  also  lower  the --length value to 18bp. If the
              smallRNA libraries are paired-end then a2 will be set to the Illumina small RNA  5'
              adapter automatically (GATCGTCGGACT) unless -a 2 had been defined explicitly.

       --consider_already_trimmed <INT>     During adapter auto-detection, the limit set by <INT>
       allows the user to

       set a threshold up to which the file is considered already adapter-trimmed. If no adapter
              sequence exceeds this threshold, no additional adapter trimming will  be  performed
              (technically, the adapter is set to '-a X'). Quality trimming is still performed as
              usual.  Default: NOT SELECTED (i.e. normal auto-detection precedence rules apply).

       --max_length <INT>      Discard reads that are longer than <INT> bp after  trimming.  This
       is only advised for

              smallRNA sequencing to remove non-small RNA sequences.

       --stringency  <INT>       Overlap  with  adapter  sequence  required  to  trim a sequence.
       Defaults to a

       very stringent setting of 1, i.e. even a single bp of overlapping sequence
              will be trimmed off from the 3' end of any read.

       -e <ERROR RATE>         Maximum allowed error rate (no. of errors divided by the length of
       the matching

              region) (default: 0.1)

       --gzip                   Compress  the  output  file  with  GZIP.  If  the input files are
       GZIP-compressed

       the output files will automatically be GZIP compressed as well. As of v0.2.8 the
              compression will take place on the fly.

       --dont_gzip             Output files won't be compressed with GZIP. This option  overrides
       --gzip.

       --length  <INT>           Discard  reads  that  became  shorter than length INT because of
       either

       quality or adapter trimming. A value of '0' effectively disables
              this behaviour. Default: 20 bp.

       For paired-end files, both reads of a read-pair need to be longer than
              <INT> bp to be printed out to validated paired-end files (see option --paired).  If
              only  one  read  became too short there is the possibility of keeping such unpaired
              single-end reads (see --retain_unpaired). Default pair-cutoff: 20 bp.

       --max_n COUNT           The total number of Ns (as integer) a read may contain  before  it
       will be removed altogether.

       In a paired-end setting, either read exceeding this limit will result in the entire
              pair being removed from the trimmed output files.

       --trim-n                 Removes  Ns  from  either  side  of  the  read.  This option does
       currently not work in RRBS mode.

       -o/--output_dir <DIR>   If specified all output will be written to this directory  instead
       of the current

              directory. If the directory doesn't exist it will be created for you.

       --no_report_file        If specified no report file will be generated.

       --suppress_warn         If specified any output to STDOUT or STDERR will be suppressed.

       --clip_R1 <int>         Instructs Trim Galore to remove <int> bp from the 5' end of read 1
       (or single-end

       reads). This may be useful if the qualities were very poor, or if there is some
              sort of unwanted bias at the 5' end. Default: OFF.

       --clip_R2 <int>         Instructs Trim Galore to remove <int> bp from the 5' end of read 2
       (paired-end reads

       only). This may be useful if the qualities were very poor, or if there is some sort
              of  unwanted bias at the 5' end. For paired-end BS-Seq, it is recommended to remove
              the first few bp because the end-repair reaction may introduce a bias  towards  low
              methylation.  Please refer to the M-bias plot section in the Bismark User Guide for
              some examples. Default: OFF.

       --three_prime_clip_R1 <int>     Instructs Trim Galore to remove <int> bp from the  3'  end
       of read 1 (or single-end

       reads) AFTER adapter/quality trimming has been performed. This may remove some unwanted
              bias  from  the 3' end that is not directly related to adapter sequence or basecall
              quality.  Default: OFF.

       --three_prime_clip_R2 <int>     Instructs Trim Galore to remove <int> bp from the  3'  end
       of read 2 AFTER

       adapter/quality trimming has been performed. This may remove some unwanted bias from
              the  3'  end  that is not directly related to adapter sequence or basecall quality.
              Default: OFF.

       --2colour/--nextseq INT This enables the option '--nextseq-trim=3'CUTOFF' within Cutadapt,
       which will set a quality

       cutoff (that is normally given with -q instead), but qualities of G bases are ignored.
              This  trimming is in common for the NextSeq- and NovaSeq-platforms, where basecalls
              without any signal are called as high-quality G bases. This  is  mutually  exlusive
              with '-q INT'.

       --path_to_cutadapt  </path/to/cutadapt>      You  may use this option to specify a path to
       the Cutadapt executable,

       e.g. /my/home/cutadapt-1.7.1/bin/cutadapt. Else it is assumed that Cutadapt is in
              the PATH.

       --basename <PREFERRED_NAME>     Use PREFERRED_NAME  as  the  basename  for  output  files,
       instead of deriving the filenames from

       the input files. Single-end data would be called PREFERRED_NAME_trimmed.fq(.gz), or
              PREFERRED_NAME_val_1.fq(.gz)  and PREFERRED_NAME_val_2.fq(.gz) for paired-end data.
              --basename only works  when  1  file  (single-end)  or  2  files  (paired-end)  are
              specified, but not for longer lists.

       -j/--cores INT          Number of cores to be used for trimming [default: 1]. For Cutadapt
       to work with multiple cores, it

       requires Python 3 as well as parallel gzip (pigz) installed on the system. The version  of
       Python used
              is detected from the shebang line of the Cutadapt executable (either 'cutadapt', or
              a specified path).  If Python 2 is detected, --cores is set to 1.  If  pigz  cannot
              be  detected  on your system, Trim Galore reverts to using gzip compression. Please
              note that gzip compression will slow down multi-core processes so much that  it  is
              hardly                   worthwhile,                   please                  see:
              https://github.com/FelixKrueger/TrimGalore/issues/16#issuecomment-458557103     for
              more info).

       Actual core usage: It should be mentioned that the actual number of cores used is a little
       convoluted.
              Assuming that Python 3 is used and pigz is installed, --cores 2 would use  2  cores
              to  read  the  input (probably not at a high usage though), 2 cores to write to the
              output (at moderately high usage), and 2 cores for Cutadapt itself +  2  additional
              cores  for  Cutadapt  (not  sure  what  they are used for) + 1 core for Trim Galore
              itself. So this can be up to 9 cores, even though most of them  won't  be  used  at
              100%  for  most of the time. Paired-end processing uses twice as many cores for the
              validation (= writing out) step.  --cores 4 would then be: 4 (read) + 4 (write) + 4
              (Cutadapt) + 2 (extra Cutadapt) +     1 (Trim Galore) = 15.

              It  seems  that  --cores  4  could  be a sweet spot, anything above has diminishing
              returns.

       SPECIFIC TRIMMING - without adapter/quality trimming

       --hardtrim5 <int>       Instead of performing adapter-/quality trimming, this option  will
       simply hard-trim sequences

       to <int> bp at the 5'-end. Once hard-trimming of files is complete, Trim Galore will exit.
              Hard-trimmed output files will end in .<int>_5prime.fq(.gz). Here is an example:

       before:
              CCTAAGGAAACAAGTACACTCCACACATGCATAAAGGAAATCAAATGTTATTTTTAAGAAAATGGAAAAT

              --hardtrim5 20: CCTAAGGAAACAAGTACACT

       --hardtrim3  <int>       Instead of performing adapter-/quality trimming, this option will
       simply hard-trim sequences

       to <int> bp at the 3'-end. Once hard-trimming of files is complete, Trim Galore will exit.
              Hard-trimmed output files will end in .<int>_3prime.fq(.gz). Here is an example:

       before:
              CCTAAGGAAACAAGTACACTCCACACATGCATAAAGGAAATCAAATGTTATTTTTAAGAAAATGGAAAAT

       --hardtrim3 20:
              TTTTTAAGAAAATGGAAAAT

       --clock                 In this mode,  reads  are  trimmed  in  a  specific  way  that  is
       currently used for the Mouse

       Epigenetic Clock (see here: Multi-tissue DNA methylation age predictor in mouse, Stubbs et
       al.,
              Genome Biology, 2017  18:68  https://doi.org/10.1186/s13059-017-1203-5).  Following
              this, Trim Galore will exit.

              In  it's  current  implementation,  the  dual-UMI  RRBS reads come in the following
              format:

       Read 1 5' UUUUUUUU CAGTA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF TACTG UUUUUUUU 3'

       Read 2 3' UUUUUUUU GTCAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF ATGAC UUUUUUUU 5'

       Where UUUUUUUU is a random 8-mer unique molecular identifier (UMI), CAGTA  is  a  constant
       region,
              and  FFFFFFF...  is  the  actual RRBS-Fragment to be sequenced. The UMIs for Read 1
              (R1) and Read 2 (R2), as well as the fixed sequences (F1 or F2), are  written  into
              the read ID and removed from the actual sequence. Here is an example:

       R1: @HWI-D00436:407:CCAETANXX:1:1101:4105:1905 1:N:0: CGATGTTT
              ATCTAGTTCAGTACGGTGTTTTCGAATTAGAAAAATATGTATAGAGGAAATAGATATAAAGGCGTATTCGTTATTG

       R2: @HWI-D00436:407:CCAETANXX:1:1101:4105:1905 3:N:0: CGATGTTT
              CAATTTTGCAGTACAAAAATAATACCTCCTCTATTTATCCAAAATCACAAAAAACCACCCACTTAACTTTCCCTAA

       R1:                    @HWI-D00436:407:CCAETANXX:1:1101:4105:1905                   1:N:0:
       CGATGTTT:R1:ATCTAGTT:R2:CAATTTTG:F1:CAGT:F2:CAGT
              CGGTGTTTTCGAATTAGAAAAATATGTATAGAGGAAATAGATATAAAGGCGTATTCGTTATTG

       R2:                   @HWI-D00436:407:CCAETANXX:1:1101:4105:1905                    3:N:0:
       CGATGTTT:R1:ATCTAGTT:R2:CAATTTTG:F1:CAGT:F2:CAGT
              CAAAAATAATACCTCCTCTATTTATCCAAAATCACAAAAAACCACCCACTTAACTTTCCCTAA

       Following    clock    trimming,    the    resulting   files   (.clock_UMI.R1.fq(.gz)   and
       .clock_UMI.R2.fq(.gz))
              should be adapter- and quality trimmed with Trim  Galore  as  usual.  In  addition,
              reads  need to be trimmed by 15bp from their 3' end to get rid of potential UMI and
              fixed sequences. The command is:

              trim_galore   --paired   --three_prime_clip_R1    15    --three_prime_clip_R2    15
              *.clock_UMI.R1.fq.gz *.clock_UMI.R2.fq.gz

       Following this, reads should be aligned with Bismark and deduplicated with UmiBam
              in  '--dual_index'  mode  (see  here: https://github.com/FelixKrueger/Umi-Grinder).
              UmiBam recognises the UMIs within  this  pattern:  R1:(ATCTAGTT):R2:(CAATTTTG):  as
              (UMI R1) and (UMI R2).

       --polyA                  This  is a new, still experimental, trimming mode to identify and
       remove poly-A tails from sequences.

       When --polyA is selected, Trim Galore attempts to identify from the first supplied  sample
       whether
              sequences contain more often a stretch of either 'AAAAAAAAAA' or 'TTTTTTTTTT'. This
              determines if Read 1 of a paired-end end file, or single-end files, are trimmed for
              PolyA  or  PolyT.  In  case  of  paired-end  sequencing,  Read2  is trimmed for the
              complementary base from the start of the reads. The auto-detection uses  a  default
              of A{20} for Read1 (3'-end trimming) and T{150} for Read2 (5'-end trimming).  These
              values may be changed manually using the options -a and -a2.

       In addition to trimming the sequences, white spaces are replaced with _ and it records  in
       the read ID
              how  many  bases  were  trimmed  so  it can later be used to identify PolyA trimmed
              sequences. This is currently done by writing tags to both the start  ("32:A:")  and
              end ("_PolyA:32") of the reads in the following example:

       @READ-ID:1:1102:22039:36996 1:N:0:CCTAATCC
              GCCTAAGGAAACAAGTACACTCCACACATGCATAAAGGAAATCAAATGTTATTTTTAAGAAAATGGAAAATAAAAACTTTATAAACACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

       @32:A:READ-ID:1:1102:22039:36996_1:N:0:CCTAATCC_PolyA:32
              GCCTAAGGAAACAAGTACACTCCACACATGCATAAAGGAAATCAAATGTTATTTTTAAGAAAATGGAAAATAAAAACTTTATAAACACC

       PLEASE NOTE: The poly-A trimming mode expects that sequences were both adapter and quality
       trimmed
              before looking for Poly-A tails, and it is the user's responsibility to  carry  out
              an initial round of trimming. The following sequence:

       1) trim_galore file.fastq.gz
              2) trim_galore --polyA file_trimmed.fq.gz 3) zcat file_trimmed_trimmed.fq.gz | grep
              -A 3 PolyA | grep -v ^-- > PolyA_trimmed.fastq

       Will 1) trim qualities and Illumina  adapter  contamination,  2)  find  and  remove  PolyA
       contamination.
              Finally,  if  desired,  3)  will  specifically  find  PolyA  trimmed sequences to a
              specific FastQ file of your choice.

       --implicon              This is a special mode of operation for paired-end data,  such  as
       required for the IMPLICON method, where a UMI sequence

              is  getting  transferred  from  the  start  of  Read 2 to the readID of both reads.
              Following this, Trim Galore will exit.

              In it's current implementation, the  UMI  carrying  reads  come  in  the  following
              format:

       Read 1 5' FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF 3'

       Read 2 3' UUUUUUUUFFFFFFFFFFFFFFFFFFFFFFFFFFFF 5'

       Where  UUUUUUUU  is a random 8-mer unique molecular identifier (UMI) and FFFFFFF... is the
       actual fragment to be
              sequenced. The UMI of Read 2 (R2) is written into the read ID  of  both  reads  and
              removed from the actual sequence.  Here is an example:

       R1: @HWI-D00436:407:CCAETANXX:1:1101:4105:1905 1:N:0: CGATGTTT
              ATCTAGTTCAGTACGGTGTTTTCGAATTAGAAAAATATGTATAGAGGAAATAGATATAAAGGCGTATTCGTTATTG

       R2: @HWI-D00436:407:CCAETANXX:1:1101:4105:1905 3:N:0: CGATGTTT
              CAATTTTGCAGTACAAAAATAATACCTCCTCTATTTATCCAAAATCACAAAAAACCACCCACTTAACTTTCCCTAA

       After --implicon trimming:
              R1: @HWI-D00436:407:CCAETANXX:1:1101:4105:1905 1:N:0: CGATGTTT:CAATTTTG

       ATCTAGTTCAGTACGGTGTTTTCGAATTAGAAAAATATGTATAGAGGAAATAGATATAAAGGCGTATTCGTTATTG
              R2: @HWI-D00436:407:CCAETANXX:1:1101:4105:1905 3:N:0: CGATGTTT:CAATTTTG

              CAGTACAAAAATAATACCTCCTCTATTTATCCAAAATCACAAAAAACCACCCACTTAACTTTCCCTAA

       RRBS-specific options (MspI digested material):

       --rrbs                   Specifies  that  the  input file was an MspI digested RRBS sample
       (recognition

       site: CCGG). Single-end or Read 1 sequences (paired-end) which were adapter-trimmed
              will have a further 2 bp removed from their 3' end.  Sequences  which  were  merely
              trimmed because of poor quality will not be shortened further. Read 2 of paired-end
              libraries will in addition have the first 2 bp removed from the 5' end (by  setting
              '--clip_r2  2').  This  is  to  avoid  using  artificial methylation calls from the
              filled-in cytosine positions close to the 3'  MspI  site  in  sequenced  fragments.
              This  option is not recommended for users of the NuGEN ovation RRBS System 1-16 kit
              (see below).

       --non_directional       Selecting this option  for  non-directional  RRBS  libraries  will
       screen

       quality-trimmed sequences for 'CAA' or 'CGA' at the start of the read
              and,  if found, removes the first two basepairs. Like with the option '--rrbs' this
              avoids using cytosine positions that were filled-in  during  the  end-repair  step.
              '--non_directional'  requires  '--rrbs'  to  be  specified  as well. Note that this
              option does not set '--clip_r2 2' in paired-end mode.

       --keep                  Keep the quality trimmed intermediate file.  Default:  off,  which
       means

       the temporary file is being deleted after adapter trimming. Only has
              an  effect  for  RRBS  samples  since  other  FastQ  files are not trimmed for poor
              qualities separately.

       Note for RRBS using the NuGEN Ovation RRBS System 1-16 kit:

       Owing to the fact that the NuGEN Ovation kit attaches  a  varying  number  of  nucleotides
       (0-3)  after  each  MspI  site  Trim  Galore should be run WITHOUT the option --rrbs. This
       trimming is accomplished in a subsequent diversity trimming  step  afterwards  (see  their
       manual).

       Note for RRBS using MseI:

       If  your  DNA material was digested with MseI (recognition motif: TTAA) instead of MspI it
       is NOT necessary to specify --rrbs or --non_directional since virtually all  reads  should
       start   with   the   sequence  'TAA',  and  this  holds  true  for  both  directional  and
       non-directional libraries. As the end-repair of 'TAA' restricted sites  does  not  involve
       any  cytosines it does not need to be treated especially. Instead, simply run Trim Galore!
       in the standard (i.e. non-RRBS) mode.

       Paired-end specific options:

       --paired                This  option  performs  length  trimming  of  quality/adapter/RRBS
       trimmed reads for

       paired-end files. To pass the validation test, both sequences of a sequence pair
              are  required  to  have  a  certain  minimum length which is governed by the option
              --length (see above). If only one read passes this length threshold the other  read
              can  be  rescued (see option --retain_unpaired). Using this option lets you discard
              too short read pairs without disturbing the  sequence-by-sequence  order  of  FastQ
              files which is required by many aligners.

       Trim Galore! expects paired-end files to be supplied in a pairwise fashion, e.g.
              file1_1.fq file1_2.fq SRR2_1.fq.gz SRR2_2.fq.gz ... .

       -t/--trim1               Trims 1 bp off every read from its 3' end. This may be needed for
       FastQ files that

       are to be aligned as paired-end data with Bowtie. This is because Bowtie (1) regards
              alignments like this:

       R1 --------------------------->
              or this:    ----------------------->  R1

       R2 <---------------------------
              <-----------------  R2

       as invalid (whenever a start/end coordinate is contained within the other read).
              NOTE: If you are planning to use Bowtie2, BWA etc. you don't need to  specify  this
              option.

       --retain_unpaired        If  only  one  of  the two paired-end reads became too short, the
       longer

       read will be written to either '.unpaired_1.fq' or '.unpaired_2.fq'
              output files. The length cutoff for unpaired single-end reads is  governed  by  the
              parameters -r1/--length_1 and -r2/--length_2. Default: OFF.

       -r1/--length_1  <INT>     Unpaired  single-end  read length cutoff needed for read 1 to be
       written to

       '.unpaired_1.fq' output file. These reads may be mapped in single-end mode.
              Default: 35 bp.

       -r2/--length_2 <INT>    Unpaired single-end read length cutoff needed for  read  2  to  be
       written to

       '.unpaired_2.fq' output file. These reads may be mapped in single-end mode.
              Default: 35 bp.

       Last modified on 07 October 2020.

AUTHOR

        This manpage was written by Nilesh Patra for the Debian distribution and
        can be used for any other usage of the program.