Provided by: igdiscover_0.11-4_all 
NAME
igdiscover - IgDiscover Documentation
IgDiscover analyzes antibody repertoires and discovers new V genes from high-throughput
sequencing reads. Heavy chains, kappa and lambda light chains are supported (to discover
VH, VK and VL genes).
IgDiscover is the result of a collaboration between the Gunilla Karlsson Hedestam group at
the Department of Microbiology, Tumor and Cell Biology at Karolinska Institutet, Sweden
and the Bioinformatics Long-Term Support facility at Science for Life Laboratory
(SciLifeLab), Sweden.
If you use IgDiscover, please cite:
Corcoran, Martin M. and Phad, Ganesh E. and Bernat, Néstor Vázquez and Stahl-Hennig,
Christiane and Sumida, Noriyuki and Persson, Mats A.A. and Martin, Marcel and
Karlsson Hedestam, Gunilla B.
Production of individualized V gene databases reveals high levels of immunoglobulin genetic
diversity.
Nature Communications 7:13642 (2016)
https://dx.doi.org/10.1038/ncomms13642
LINKS
• Documentation
• Source code
• Report an issue
• Project page on PyPI (Python package index)
[image]
INSTALLATION
IgDiscover is written in Python 3 and is developed on Linux. The tool also runs on macOS,
but is not as well tested on that platform.
For installation on either system, we recommend that you follow the instructions below,
which will first explain how to install the Conda package manager. IgDiscover is available
as a Conda-package from the bioconda channel. Using Conda will make the installation easy
because all dependencies are also available as Conda packages and can thus be installed
automatically along with IgDiscover.
There are also non-Conda installation instructions if you cannot use Conda.
Installing IgDiscover with Conda
1. Install Conda by following the conda installation instructions as appropriate for your
system. You will need to choose between a “Miniconda” and “Anaconda” installation. We
recommend Miniconda as the download is smaller. If you are in a hurry, these two
commands are usually sufficient to install Miniconda on Linux (read the linked document
for macOS instructions):
wget https://repo.continuum.io/miniconda/Miniconda3-latest-Linux-x86_64.sh
bash Miniconda3-latest-Linux-x86_64.sh
When the installer asks you about modifying the PATH in your .bashrc file, answer yes.
2. Close the terminal window and open a new one. Then test whether conda is installed
correctly by running
conda --version
If you see the conda version number, it worked.
3. Set up Conda so that it can access the bioconda channel. For that, follow the
instructions on the bioconda website or simply run these commands:
conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge
4. Install IgDiscover with this command:
conda create -n igdiscover igdiscover
This will create a new so-called “environment” for IgDiscover (retry if it fails).
Whenever you want to run IgDiscover, you will need to activate the environment with
this command:
source activate igdiscover
5. Make sure you have activated the igdiscover environment. Then test whether IgDiscover
is correctly installed with this command:
igdiscover --version
If you see the version number of IgDiscover, it worked! If an error message appears
that says "The 'networkx' distribution was not found and is required by snakemake",
install networkx manually with:
pip install networkx==2.1
Then retry to check the igdiscover version.
6. You can now run IgDiscover on the test data set to familiarize yourself with how it
works.
Troubleshooting on Linux
If you use zsh instead of bash (applies to Bio-Linux, for example), the $PATH environment
variable will not be setup correctly by the Conda installer. The miniconda installer adds
a line export PATH=... to the to the end of your /home/your-user-name/.bashrc file. Copy
that line from the file and add it to the end of the file /home/your-user-name/.zshrc
instead.
Alternatively, change your default shell to bash by running chsh -s /bin/bash.
If you use conda and see an error that includes something like this:
ImportError: .../.local/lib/python3.5/site-packages/sqt/_helpers.cpython-35m-x86_64-linux-gnu.so: undefined symbol: PyFPE_jbuf
Or you see any error that mentions a .local/ directory, then a previous installation of
IgDiscover is interfering with the conda installation.
The easiest way to solve this problem is to delete the directory .local/ in your home
directory, see also how to remove IgDiscover from a Linux system.
Troubleshooting on macOS
If you get the error
ValueError: unknown locale: UTF-8
Then follow these instructions.
Development version
To install IgDiscover directly from the most recent source code, read the developer
installation instructions.
MANUAL INSTALLATION
IgDiscover requires quite a few other software tools that are not included in most Linux
distributions (or mac OS) and which are also not available from the Python packaging index
(PyPI) because they are not Python tools. If you do not use the recommended simple
installation instructions via Conda, you need to install those non-Python dependencies
manually. Regular Python dependencies are automatically pulled in when IgDiscover itself
is installed in the last step with the pip install command. The instructions below are
written for Linux and require modifications if you want to try this on OS X.
NOTE:
We recommend the much simpler installation via Conda instead of using the instructions
in this section.
Install non-Python dependencies
The dependencies are: MUSCLE, IgBLAST, PEAR, and -- optionally -- flash.
1. Install Python 3.5 or newer. It most likely is already installed on your system, but in
Debian/Ubuntu, you can get it with
sudo apt-get install python3
2. Create the directory where binaries will be installed. We assume $HOME/.local/bin here,
but this can be anywhere as long as they are in your $PATH.
mkdir -p ~/.local/bin
Add this line to the end of your ~/.bashrc file:
export PATH=$HOME/.local/bin:$PATH
Then either start a new shell or run source ~/.bashrc to get the changes.
3. Install MUSCLE. This is available as a package in Ubuntu:
sudo apt-get install muscle
If your distribution does not have a 'muscle' package or if you are not allowed to run
sudo:
wget -O - http://www.drive5.com/muscle/downloads3.8.31/muscle3.8.31_i86linux64.tar.gz | tar xz
mv muscle3.8.31_i86linux64 ~/.local/bin/
4. Install PEAR:
wget http://sco.h-its.org/exelixis/web/software/pear/files/pear-0.9.6-bin-64.tar.gz
tar xvf pear-0.9.6-bin-64.tar.gz
mv pear-0.9.6-bin-64/pear-0.9.6-bin-64 ~/.local/bin/pear
5. Install IgBLAST:
wget ftp://ftp.ncbi.nih.gov/blast/executables/igblast/release/1.4.0/ncbi-igblast-1.4.0-x64-linux.tar.gz
tar xvf ncbi-igblast-1.4.0-x64-linux.tar.gz
mv ncbi-igblast-1.4.0/bin/igblast? ~/.local/bin/
IgBLAST requires some data files that must be downloaded separately. The following
commands put the files into ~/.local/igdata:
mkdir ~/.local/igdata
cd ~/.local/igdata
wget -r -nH --cut-dirs=4 ftp://ftp.ncbi.nih.gov/blast/executables/igblast/release/internal_data
wget -r -nH --cut-dirs=4 ftp://ftp.ncbi.nih.gov/blast/executables/igblast/release/database/
wget -r -nH --cut-dirs=4 ftp://ftp.ncbi.nih.gov/blast/executables/igblast/release/optional_file/
Also, you must set the $IGDATA environment variable to point to the directory with data
files. Add this line to your ~/.bashrc:
export IGDATA=$HOME/.local/igdata
Then run source ~/.bashrc to get the changes.
7. Optionally, install flash:
wget -O FLASH-1.2.11.tar.gz http://sourceforge.net/projects/flashpage/files/FLASH-1.2.11.tar.gz/download
tar xf FLASH-1.2.11.tar.gz
cd FLASH-1.2.11
make
mv flash ~/.local/bin/
Install IgDiscover
Install IgDiscover with the Python package manager pip, which will download and install
IgDiscover and its dependencies:
pip3 install --user igdiscover
Both commands also install all remaining dependencies. The --user option instructs both
commands to install everything into $HOME/.local.
Finally, check the installation with
igdiscover --version
and you should see the version number of IgDiscover.
You should now run IgDiscover on the test data set.
TEST DATA SET
After installing IgDiscover, you should run it once on a small test data that we provide,
both to test your installation and to familiarize yourself with running the program.
1. Download und unpack the test data set (version 0.5). To do this from the command-line,
use these commands:
wget https://bitbucket.org/igdiscover/testdata/downloads/igdiscover-testdata-0.5.tar.gz
tar xvf igdiscover-testdata-0.5.tar.gz
The test data set contains some paired-end reads from human IgM heavy chain dataset
ERR1760498 and a database of IGHV, IGHD, IGHJ sequences based on Ensembl annotations.
You should use a database of higher quality for your own experiments.
2. Initialize the IgDiscover pipeline directory:
igdiscover init --db igdiscover-testdata/database/ --reads igdiscover-testdata/reads.1.fastq.gz discovertest
The name discovertest is the name of the pipeline directory that will be created. Note
that only the path to the first reads file needs to be given. The second file is found
automatically. There may be a couple of messages “Skipping 'x' because it contains the
same sequence as 'y'”, which you can ignore.
The command will have printed a message telling you that the pipeline directory has
been initialized, that you should edit the configuration file, and how to actually run
IgDiscover after that.
3. The generated igdiscover.yaml configuration file does not actually need to be edited
for the test dataset, but you may still want to have a read through it as you will need
to do so for you own data. You may want to do this while the pipeline is running in the
next step. The configuration is in YAML format. When editing the file, just follow the
way it is already structured.
4. Run the analysis. To do so, change into the pipeline directory and run this command:
cd discovertest && igdiscover run
On this small dataset, running the pipeline should take not more than about 5 minutes.
5. Finally, inspect the results in the discovertest/iteration-01 or discovertest/final
directories. The discovered V genes and extra information are listed in
discovertest/iteration-01/new_V_germline.tab. Discovered J genes are in
discovertest/iteration-01/new_J.tab. There are also corresponding .fasta files with the
sequences only.
See the explanation of final result files.
Other test data sets
ENA project PRJEB15295 contains the data for our Nature Communications paper from 2016, in
particular ERR1760498, which is the data for the human “H1” sample (multiplex PCR, IgM
heavy chain).
Data used for testing TCR detection (human, RACE): SRR2905677 and SRR2905710.
USER GUIDE
Overview
IgDiscover works on a single library at a time. It works within an “analysis directory”
for the library, which contains all intermediate and result files.
To start an analysis, you need:
1. A FASTA or FASTQ file with single-end reads or two FASTQ files with paired-end reads
(also, the files must be gzip-compressed)
2. A database of V/D/J genes (three FASTA files named V.fasta, D.fasta, J.fasta)
3. A configuration file that describes the library
If you do not have a V/D/J database, yet, you may want to read the section about how to
obtain V/D/J sequences.
To run an analysis, proceed as follows.
NOTE:
If you are on macOS, it may be necessary to run export SHELL=/bin/bash before
continuing.
1. Create and initialize the analysis directory.
First, pick a name for your analysis. We will use myexperiment in the following. Then
run igdiscover init:
igdiscover init myexperiment
A dialog will appear and ask for the file with the first (forward) reads. Find your
compressed FASTQ file that contains them and select it. Typical file names may be
Library1_S1_L001_R1_001.fastq.gz or mylibrary.1.fastq.gz. You do not need to choose
the second read file! It is found automatically.
Next, choose the directory with your database. The directory must contain the three
files V.fasta, D.fasta, J.fasta. These files contain the V, D, J gene sequences,
respectively. Even if have have only light chains in your data, a D.fasta file needs
to be provided, just use one with the heavy chain D gene sequences.
If you do not want a graphical user interface, use the two command-line parameters --db
and --reads1 to provide this information instead:
igdiscover init --db path/to/my/database/ --reads1 mylibrary.1.fastq.gz myexperiment
Again, the second reads file will be found automatically. Use --single-reads instead
of --reads1 if you have single-end reads or a dataset with already merged reads. For
--single-reads, a FASTA file (not only FASTQ) is also allowed. In any case, an
analysis directory named myexperiment will have been created.
2. Adjust the configuration file
The previous step created a configuration file named myexperiment/igdiscover.yaml,
which you may need to adjust. In particular, the number of discovery rounds is set to 3
by default, which takes a long time. Reducing this to 2 or even 1 often works just as
well.
3. Run the analysis
Change into the newly created analysis directory and run the analysis:
igdiscover run
Depending on the size of your library, your computer, and the number of iterations,
this will now take from a few hours to a day. See the running IgDiscover section for
more fine-grained control over what to run and how to resume the process if something
failed.
Obtaining a V/D/J database
We use the term “database” to refer to three FASTA files that contain the sequences for
the V, D and J genes. IMGT provides sequences for download. For discovering new VH
genes, for example, you need to get the IGHV, IGHD and IGHJ files of your species. As
IgDiscover uses this only as a starting point, using a similar species will also work.
When using an IMGT database, it is very important to change the long IMGT sequence headers
to short headers as IgBLAST does not accept the long headers. We recommend using the
program edit_imgt_file.pl. If you installed IgDiscover from Conda, the script is already
installed and you can run it by typing the name. It is also available on the IgBlast FTP
site.
Run it for all three downloaded files, and then rename files appropritely to make sure
that they named V.fasta, D.fasta and J.fasta.
You always need a file with D genes even if you analyze light chains.
In case you have used IgBLAST previously, note that there is no need to run the
makeblastdb tool as IgDiscover will do that for you.
Input data requirements
Paired-end or single-end data
IgDiscover can process input data of three different types:
• Paired-end reads in gzipped FASTQ format,
• Single-end reads in gzipped FASTQ format,
• Single-end reads in gzipped FASTA format.
IgDiscover was tested mainly on paired-end Illumina MiSeq reads (2x300bp), but it can also
handle 454 and Ion Torrent data.
Depending on the input file type, use a variant of one of the following commands to
initialize the analysis directory:
igdiscover init --single-reads=file.fasta.gz --database=my-database-dir/ myexperiment
igdiscover init --reads1=file.1.fasta.gz --database=my-database-dir/ myexperiment
igdiscover init --reads1=file.1.fastq.gz --database=my-database-dir/ myexperiment
Read layout
Paired-end reads are first merged and then processed in the same way as single-end reads.
Reads that could not be merged are discarded. Single-end reads and merged paired-end reads
are expected to follow this structure (from 5' to 3'):
• The forward primer sequence. This is optional.
• A random barcode (molecular identifier). This is optional. Set the configuration option
barcode_length_5p to 0 if you don’t have random barcodes or if you don’t want the
program to use them.
• Optionally, a run of G nucleotides. This is an artifact of the RACE protocol (Rapid
amplification of cDNA ends). If you have this, set race_g to true in the configuration
file.
• 5' UTR
• Leader
• Re-arranged V, D and J gene sequences for heavy chains; only V and J for light chains
• An optional random barcode. Set the configuration option barcode_length_3p to the length
of this barcode. You can currently not have both a 5' and a 3' barcode.
• The reverse primer. This is optional.
We use IgBLAST to detect the location of the V, D, J genes through the igdiscover igblast
subcommand. The G nucleotides after the barcode are split off if the configuration
specifies race_g: true. The leader sequence is detected by looking for a start codon near
60 bp upstream of the start of the V gene match.
Configuration
The igdiscover init command creates a configuration file igdiscover.yaml in the analysis
directory. To configure your analysis, change that file with a text editor before running
the analysis with igdiscover run.
The syntax should be mostly self-explanatory. The file is in YAML format, but you will
not need to learn that. Just follow the examples given in the file. A few rules that may
be good to know are the following ones:
1. Lines starting with the # symbol are comments (they are ignored)
2. A configuration option that is meant to be switched on or off will say something like
stranded: false if it is off. Change this to stranded: true to switch the option on
(and vice versa).
3. The primer sequences are given as a list, and must be written in a certain way - one
sequence per line, and a - (dash) in front, like so:
forward_primers:
- ACGTACGTACGT
- AACCGGTTAACC
Even if you have only one primer sequence, you still need to use this syntax.
To find out what the configuration options achieve, see the explanations in the
configuration file itself.
The main parameters parameters that may require adjusting are the following.
The iterations option sets the number of rounds of V gene discovery that will be
performed. By default, three iterations are run. Even with a very restricted starting V
database (for example with only a single V gene sequence), this is usually sufficient to
identify most novel germline sequences.
When the starting database is more complete, for example, when analyzing a human IgM
library with the current IMGT heavy chain database, a single iteration may be sufficient
to produce an individualized database.
If you do not want to discover any new genes and only want to produce an expression
profile, for example, then use iterations: 0.
The ignore_j option should be set to true when producing a V gene database for a species
where J sequences are unknown:
ignore_j: true
Setting the parameters stranded, forward_primers and reverse_primers to the correct values
can be used to remove 5' and 3' primers from the sequences. Doing this is not strictly
necessary for IgDiscover. It is simplest if you do not specify any primer sequences.
Pregermline and germline filter criteria
This provides IgDiscover with stringency requirements for V gene discovery that enable the
program to filter out false positives. Usually the ”pregermline filter” can be used in the
default mode since all these sequences will be subsequently passed to the higher
stringency ”germline filter” where the criteria are set to maximize stringency. Here is
how it looks in the configuration file:
pre_germline_filter:
unique_cdr3s: 2 # Minimum number of unique CDR3s (within exact matches)
unique_js: 2 # Minimum number of unique J genes (within exact matches)
check_motifs: false # Check whether 5' end starts with known motif
whitelist: true # Add database sequences to the whitelist
cluster_size: 0 # Minimum number of sequences assigned to cluster
differences: 0 # Merge sequences if they have at most this number of differences
allow_stop: true # Whether to allow non-productive sequences containing stop codons
cross_mapping_ratio: 0.02 # Threshold for removal of cross-mapping artifacts (set to 0 to disable)
allele_ratio: 0.1 # Required minimum ratio between alleles of a single gene
# Filtering criteria applied to candidate sequences in the last iteration.
# These should be more strict than the pre_germline_filter criteria.
#
germline_filter:
unique_cdr3s: 5 # Minimum number of unique CDR3s (within exact matches)
unique_js: 3 # Minimum number of unique J genes (within exact matches)
check_motifs: false # Check whether 5' end starts with known motif
whitelist: true # Add database sequences to the whitelist
cluster_size: 100 # Minimum number of sequences assigned to cluster
differences: 0 # Merge sequences if they have at most this number of differences
allow_stop: false # Whether to allow non-productive sequences containing stop codons
cross_mapping_ratio: 0.02 # Threshold for removal of cross-mapping artifacts (set to 0 to disable)
allele_ratio: 0.1 # Required minimum ratio between alleles of a single gene
Factors that affect germline discovery include library source (IgM vs IgK, IgL or IgG)
library size, sequence error rate and individual genomic factors (for example the number
of J segments present in an individual).
In general, setting a higher cutoff of unique_cdr3s and unique_js will minimize the number
of false positives in the output. Example:
unique_cdr3s: 10 # Minimum number of unique CDR3s (within exact matches)
unique_js: 4 # Minimum number of unique J genes (within exact matches)
If the differences parameter is set to a value higher than 0, the germline filter inspects
clusters of sequences that are closely related (when the edit distance between them is at
most differences) and retains only the most common sequence of each cluster. Previously,
we believed this would removes some false positives due to accumulated random sequence
errors of highly expressed alleles that otherwise would pass the cutoff criteria. However,
we found out that we miss true positives, in particular if there are two alleles in the
sample that differ in only a single nucleotide. We have now implemented other measures to
avoid false positives and recommend against setting the differences to something other
than 0.
Read also about the cross mapping, for which germline filtering corrects, and about the
germline filters.
Changed in version The: default for the differences setting was changed from 1 to 0.
Running IgDiscover
Resuming failed runs
The command igdiscover run, which is used to start the pipeline, can also be used to
resume execution if there was an interruption (a transient failure). Reasons for
interruptions might be:
• Ctrl+C was pressed on the keyboard
• A full harddisk
• If running on a cluster, the program may have been terminated because it exceeded its
allocated time
• Too little RAM
• Power loss
To resume execution after you have fixed the problem, go to the analysis directory and run
igdiscover run again. It will skip the steps that have already finished successfully.
This capability comes from the workflow management system snakemake, on which igdiscover
run is based. Snakemake will determine automatically which steps need to be re-run in
order to get to a full result and then run only those.
Alterations to the configuration file after an interruption are possible, but affect only
steps that have not already finished successfully. For example, assume you interrupted a
run with Ctrl+C after it is already past the step in which barcodes are removed. Then,
even if you change the barcode length in the configuration, the barcode removal step will
not be re-run when you resume the pipeline and the previous barcode length is in effect.
See also the next section.
Changing parameters and re-running parts of the pipeline
When you experiment with parameters in the igdiscover.yaml file, such as germline
filtering criteria, you do not need to re-run the entire pipeline from the beginning, but
can re-use the results that already exist. This can save a lot of processing time, in
particular when you avoid re-running IgBLAST in this way.
As described in the previous section, igdiscover run automatically figures out which files
need to be re-created if a run was interrupted. Unfortunately, this mechanism is currently
not smart enough to also look for changes in the igdiscover.yaml file. Thus, if the full
pipeline has finished successfully, then re-running igdiscover run will just print the
message Nothing to be done. even after you have changed the configuration file.
You will therefore need to know yourself which file you want to regenerate. Then follow
the following steps. Note that these will remove parts of the existing results, and if you
need to keep them, make a copy of your analysis directory first.
1. Change the configuration setting.
2. Delete the file that needs to be re-generated. Assume it is filename
3. Run igdiscover run filename to re-create the file. Only that file will be created, not
the ones that usually would be created afterwards.
4. Optionally, run igdiscover run (without a file name this time) to update the remaining
files (those that depend on the file that was just updated).
For example, assume you want to modify some germline filtering setting and then re-run the
pipeline. Change the setting in your igdiscover.yaml, then run these commands:
rm iteration-01/new_V_germline.tab
igdiscover run iteration-01/new_V_germline.tab
The above will have regenerated the iteration-01/new_V_germline.tab file and also the
iteration-01/new_V_germline.fasta file since they are generated by the same script. If you
want to update any other files, then also run
igdiscover run
The analysis directory
IgDiscover writes all intermediate files, the final V gene database, statistics and plots
into the analysis directory that was created with igdiscover init. Inside that directory,
there is a final/ subdirectory that contains the analysis results.
These are the files and subdirectories that can be found in the analysis directory.
Subdirectories are described in detail below.
igdiscover.yaml
The configuration file. Make sure to adjust this to your needs as described above.
reads.1.fastq.gz, reads.2.fastq.gz
Symbolic links to the raw paired-end reads.
database/
The input V/D/J database (as three FASTA files). The files are a copy of the ones
you selected when running igdiscover init.
reads/ Processed reads (merged, de-duplicated etc.)
iteration-xx/
Iteration-specific analysis directory, where “xx” is a number starting from 01.
Each iteration is run in one of these directories. The first iteration (in
iteration-01) uses the original input database, which is also found in the
database/ directory. The database is updated and then used as input for the next
iteration.
final/ After the last iteration, IgBLAST is run again on the input sequences, but using
the final database (the one created in the very last iteration). This directory
contains all the results, such as plots of the repertoire profiles. If you set the
number of iterations to 0 in the configuration file, this directory is the only one
that is created.
Final results
Final results are found in the final/ subdirectory of the analysis directory.
final/database/(V,D,J).fasta
These three files represent the final, individualized V/D/J database found by
IgDiscover. The D and J files are copies of the original starting database; they
are not updated by IgDiscover.
final/dendrogram_(V,D,J).pdf
These three PDF files contain dendrograms of the V, D and J sequences in the
individualized database.
final/assigned.tab.gz
V/D/J gene assignments and other information for each sequence. The file is
created by parsing the IgBLAST output in the igblast.txt.gz file. This is a table
that contains one row for each input sequence. See below for a detailed
description of the columns.
final/filtered.tab.gz
Filtered V/D/J gene assignments. This is the same as the assigned.tab file
mentioned above, but with low-quality assignments filtered out. Run igdiscover
filter --help to see the filtering criteria.
final/expressed_(V,D,J).tab, final/expressed_(V,D,J).pdf
The V, D and J gene expression counts. Some assignments are filtered out to reduce
artifacts. In particular, an allele-ratio filter of 10% is applied. For D genes,
only those with an E-value of at most 1E-4 and a coverage of at least 70% are
counted. See also the help for the igdiscover count subcommand, which is used to
create these files.
The .tab file contains the counts as a table, while the PDF file contains a plot of
the same values.
These tables also exist in the iteration-specific directories (iteration-xx). For
those, note that the numbers do not include the genes that were discovered in that
iteration. For example, iteration-01/expressed_V.tab shows only expression counts
of the V genes in the starting database.
final/errorhistograms.pdf
A PDF with one page per V gene/allele. Each page shows a histogram of the
percentage differences for that gene.
final/clusterplots/
This is a directory that contains one PNG file for each discovered gene/allele.
Each image shows a clusterplot of all the sequences assigned to that gene. Note
that the shown clusterplots are by default restricted to showing only at most 300
sequences, while the actual clustering used by IgDiscover uses 1000 sequences.
If you are interested in the results of each iteration, you can inspect the iteration-xx/
directories. They are structured in the same way as the final/ subdirectory, except that
the results are based on the intermediate databases of that iteration. They also contain
the following additional files.
iteration-xx/candidates.tab
A table with candidate novel V alleles (or genes). This is a list of sequences
found through the windowing strategy or linkage cluster analysis, as discussed in
our paper. See the full description of candidates.tab.
iteration-xx/read_names_map.tab
For each candidate novel V allele listed in candidates.tab, this file contains one
row that lists which sequences went into generating this candidate. Only the exact
matches are listed, that is, the number of listed sequence names should be equal to
the value in the exact column. Each line in this file contains tab-separated
values. The first is name of the candidate, the others are the names of the
sequences. Some of these sequences may be consensus sequences if barcode grouping
was enabled, so in that case, this will not be a read name.
iteration-xx/new_V_germline.fasta, iteration-xx/new_V_pregermline.fasta
The discovered list of V genes for this iteration. The file is created from the
candidates.tab file by applying either the germline or pre-germline filter. The
file resulting from application of the germline filter is used in the last
iteration only. The file resulting from application of the pre-germline filter is
used in earlier iterations.
iteration-xx/annotated_V_germline.tab, iteration-xx/annotated_V_pregermline.tab
A version of the candidates.tab file that is annotated with extra columns that
describe why a candidate was filtered out. See the description of this file.
Other files
For completeness, here is a description of the files in the reads/ and stats/ directories.
They are created during pre-processing and are not iteration specific.
reads/1-limited.1.fastq.gz, reads/1-limited.1.fastq.gz
Input reads file limited to the first N entries. This is just a symbolic link to
the input file if the limit configuration option is not set.
reads/2-merged.fastq.gz
Reads merged with PEAR or FLASH
reads/3-forward-primer-trimmed.fastq.gz
Merged reads with 5' primer sequences removed. (This file is automatically removed
when it is not needed anymore.)
reads/4-trimmed.fastq.gz
Merged reads with 5' and 3' primer sequences removed.
reads/5-filtered.fasta
Merged, primer-trimmed sequences converted to FASTA, and too short sequences
removed. (This file is automatically removed when it is not needed anymore.)
reads/sequences.fasta.gz
Fully pre-processed sequences. That is, filtered sequences without duplicates
(using VSEARCH)
stats/reads.txt
Statistics of pre-processed sequences.
stats/readlengths.txt, stats/readlengths.pdf
Histogram of the lengths of pre-processed sequences (created from
reads/sequences.fasta)
Format of output files
assigned.tab.gz
This file is a gzip-compressed table with tab-separated values. It is created by the
igdiscover igblast subcommand and is the result of parsing raw output from IgBLAST. It
contains a few additional columns that do not come directly from IgBLAST. In particular,
the CDR3 sequence is detected, the sequence before the V gene match is split into UTR and
leader, and the RACE-specific run of G nucleotides is also detected. The first row is a
header row with column names. Each subsequent row describes the IgBLAST results for a
single pre-processed input sequence.
Note: This file is typically quite large. LibreOffice can open the file directly (even
though it is compressed), but make sure you have enough RAM.
Columns:
count How many copies of input sequence this query sequence represents. Copied from the
;size=3; entry in the FASTA header field that is added by VSEARCH
-derep_fulllength.
V_gene, D_gene, J_gene
V/D/J gene match for the query sequence
stop whether the sequence contains a stop codon (either “yes” or “no”)
productive
V_covered, D_covered, J_covered
percentage of bases of the reference gene that is covered by the bases of the query
sequence
V_evalue, D_evalue, J_evalue
E-value of V/D/J hit
FR1_SHM, CDR1_SHM, FR2_SHM, CDR2_SHM, FR3_SHM, V_SHM, J_SHM
rate of somatic hypermutation (actually, an error rate)
V_errors, J_errors
Absolute number of errors (differences) in the V and J gene match
UTR Sequence of the 5' UTR (the part before the V gene match up to, but not including,
the start codon)
leader Leader sequence (the part between UTR and the V gene match)
CDR1_nt, CDR1_aa, CDR2_nt, CDR2_aa, CDR3_nt, CDR3_aa
nucleotide and amino acid sequence of CDR1/2/3
V_nt, V_aa
Nucleotide and amino acid sequence of V gene match
V_CDR3_start
Start coordinate of CDR3 within V_nt. Set to zero if no CDR3 was detected.
Comparisons involving the V gene ignore those V bases that are part of the CDR3.
V_end, VD_junction, D_region, DJ_junction, J_start
nucleotide sequences for various match regions
name, barcode, race_G, genomic_sequence
see the following explanation
The UTR, leader, barcode, race_G and genomic_sequence columns are filled in the following
way.
1. Split the 5' end barcode from the sequence (if barcode length is zero, this will be
empty), put it in the barcode column.
2. Remove the initial run of G bases from the remaining sequence, put that in the race_G
column.
3. The remainder is put into the genomic_sequence column.
4. If there is a V gene match, take the sequence before it and split it up in the
following way. Search for the start codon and write the part before it into the UTR
column. Write the part starting with the start column into the leader column.
filtered.tab.gz
This table is the same as the assigned.tab.gz table, except that rows containing
low-quality matches have been filtered out. Rows fulfilling any of the following criteria
are filtered:
• The J gene was not assigned
• A stop was codon found
• The V gene coverage is less than 90%
• The J gene coverage is less than 60%
• The V gene E-value is greater than 10-3
candidates.tab
This table contains the candidates for novel V genes found by the discover subcommand. As
the other files, it is a text file in tab-separated values format, with the first row
containing the column headings. It can be opened directly in LibreOffice, for example.
Candidates are found by inspecting all the sequences assigned to a database gene, and
clustering them in multiple ways. The candidate sequences are found by computing a
consensus from each found cluster.
Each row describes a single candidate, but possibly multiple clusters. If there are
multiple clusters from a single gene that lead to the same consensus sequence, then they
get only one row. The cluster column lists the source clusters for the given sequence.
Duplicate sequences can still occur when two different genes lead to identical consensus
sequences. (These duplicated sequences are merged by the germline filters.)
Below, we use the term cluster set to refer to all the sequences that are in any of the
listed clusters.
Some clusters lead to ambiguous consensus sequences (those that include N bases). These
have already been filtered out.
name The name of the candidate gene. See novel gene names.
source The original database gene to which the sequences from this row were originally
assigned. All candidates coming from the same source gene are grouped together.
chain Chain type: VH for heavy, VK for light chain lambda, VL for light chain kappa
cluster
From which type of cluster or clusters the consensus was computed. If there are
multiple clusters that give rise to the same consensus sequence, they are all
listed here, separated by semicolon. A cluster name such as 2-4 is for a
percentage difference window: Such a cluster consists of all sequences assigned to
the source gene that have a percentage difference to it between 2 and 4 percent.
A cluster name such as cl3 describes a cluster generated through linkage cluster
analysis. The clusters are simply named cl1, cl2, cl3 etc. If any cluster number
seems to be missing (such as when cl1 and cl3 occur, but not cl2), then this means
that the cluster led to an ambiguous consensus sequence that has been filtered out.
Since the cl clusters are created from a random subsample of the data (in order to
keep computation time down), they are never larger than the size of the subsample
(currently 1000).
The name db represents a cluster that is identical to the database sequence. If no
actual cluster corresponding to the database sequence is found, but the database
sequence is expressed, a db cluster is inserted artificially in order to make sure
that the sequence is not lost. The cluster name all represents the set of all
sequences assigned to the source gene. This means that an unambiguous consensus
could be computed from all the sequences. Typically, this happens during later
iterations when there are no more novel sequences among the sequences assigned to
the database gene.
cluster_size
The number of sequences from which the consensus was computed. Equivalently, the
size of the cluster set (all clusters described in this row). Sequences that are
in multiple clusters at the same time are counted only once.
Js The number of unique J genes associated with the sequences in the cluster set.
Consensus sequences are computed only from V gene sequences, but each V gene
sequence is part of a full V/D/J sequence. We therefore know for each V sequence
which J gene it was found with. This number says how many different J genes were
found for all sequences that the consensus in this row was computed from.
CDR3s The number of unique CDR3 sequences associated with the sequences in the cluster
set. See also the description for the Js column. This number says how many
different CDR3 sequences were found for all sequences that the consensus in this
row was computed from.
exact The number of exact occurrences of the consensus sequence among all sequences
assigned to the source gene, ignoring the 3' junction region.
To clarify: While the consensus sequence is computed only from a subset of
sequences assigned to a source gene, all sequences assigned to the source gene are
searched for exact occurrences of that consensus sequence.
When comparing sequences, they are first truncated at the 3' end by removing those
(typically 8) bases that correspond to the CDR3 region.
barcodes_exact
How many unique barcode sequences were used by the sequences in the set of exact
sequences (described above).
Ds_exact
How many unique D genes were used by the sequences in the set of exact sequences
(described above). Only those D gene assignments are included in this count for
which the number of errors is zero, the E-value is at most a given threshold, and
for which the number of covered bases is at least a given percentage.
Js_exact
How many unique J genes were used by the sequences in the set of exact sequences
(described above).
CDR3s_exact
How many unique CDR3 sequences were used by the sequences in the set of exact
sequences (described above).
clonotypes
The estimated number of clonotypes within the set of exact sequences (which is
described above). The value is computed by clustering the unique CDR3 sequences
associated with all exact occurrences, allowing up to six differences (mismatches,
insertions, deletions) and then counting the number of resulting clusters.
database_diff
The number of differences between the consensus sequence and the sequence of the
source gene. (Given as edit distance, that is insertion, deletion, mismatch count
as one difference each.)
has_stop
Indicates whether the consensus sequence contains a stop codon.
looks_like_V
Whether the consensus sequence “looks like” a true V gene (1 if yes, 0 if no).
Currently, this checks whether the 5' end of the sequence matches a known V gene
motif.
CDR3_start
Where the CDR3 starts within the discovered V gene sequence. This uses the most
common CDR3 start location among the sequences from which this consensus is
derived.
consensus
The consensus sequence itself.
The igdiscover discover command can also be run by hand with other parameters, in which
case additional columns may appear.
N_bases
Number of N bases in the consensus
annotated_V_*.tab
The two files annotated_V_germline.tab and annotated_V_pregermline.tab are copies of the
candidates.tab file with two extra columns that show why a candidate was filtered in the
germline and pre-germline filtering steps. The two columns are:
• is_filtered – This is a number that indicates how many filtering criteria exclude
this candidate apply.
• why_filtered – This is a semicolon-separated list of filtering reasons.
The following values can occur in the why_filtered column:
too_low_dbdiff
The number of differences between this candidate and the database is lower than the
required number.
too_many_N_bases
The candidate contains too many N nucleotide wildcard characters.
too_low_CDR3s_exact
The CDR3s_exact value for this candidate is lower than required.
too_high_CDR3_shared_ratio
The CDR3_shared_ratio is higher than the configured threshold.
too_low_Js_exact
The Js_exact value is lower than the configured threshold.
has_stop
The filter configuration disallows stop codons, but this candidate has one and is
not whitelisted.
too_low_cluster_size
The cluster_size of this candidate is lower than the configured threshold, and the
candidate is not whitelisted.
is_duplicate
A filtering criterion not listed above applies to this candidate. This covers all
the filters that need to compare candidates to each other: cross-mapping ratio,
clonotype allele ratio, exact ratio, Ds_exact ratio.
Names for discovered genes
Each gene discovered by IgDiscover gets a unique name such as “VH4.11_S1234”. The
“VH4.11” is the name of the database gene to which the novel V gene was initially
assigned. The number 1234 is derived from the nucleotide sequence of the novel gene. That
is, if you discover the same sequence in two different runs of the IgDiscover, or just in
different iterations, the number will be the same. This may help when manually inspecting
results.
Be aware that you still need to check the sequence itself since even different sequences
can sometimes lead to the same number (a “hash collision”).
The _S1234 suffixes do not accumulate. Before IgDiscover adds the suffix in an iteration,
it removes the suffix if it already exists.
Subcommands
The igdiscover program has multiple subcommands. You should already be familiar with the
two commands init and run. Each subcommand comes with its own help page that shows how to
use that subcommand. Run the command with the --help option to see the help. For example,
igdiscover run --help
shows the help for the run subcommand.
The following additional subcommands may be useful for further analysis.
commonv
Find common V genes between two different antibody libraries
upstream
Cluster upstream sequences (UTR and leader) for each gene
dendrogram
Draw a dendrogram of sequences in a FASTA file.
rename Rename sequences in a target FASTA file using a template FASTA file
union Compute union of sequences in multiple FASTA files
The following subcommands are used internally, and listed here for completeness.
filter Filter a table with IgBLAST results
count Count and plot V, D, J gene usage
group Group sequences by barcode and V/J assignment and print each group’s consensus
(unused in IgDiscover)
germlinefilter
Create new V gene database from V gene candidates using the germline and
pre-germline filter criteria.
discover
Discover candidate new V genes within a single antibody library
clusterplot
For each V gene, plot a clustermap of the sequences assigned to it
errorplot
Plot histograms of differences to reference V gene
Germline and pre-germline filtering
V gene sequences found by the clustering step of the program (the discover subcommand) are
stored in the candidates.tab file. The entries are “candidates” because many of these will
be PCR or other artifacts and therefore do not represent true novel V genes. The germline
and pre-germline filters take care of removing artifacts. They germline filter is the
“real” filter and used only in the last iteration in order to obtain the final gene
database. The pre-germline filter is less strict and used in all the earlier iterations.
The germline filters are implemented in the igdiscover germlinefilter subcommand. It
performs the following filtering and processing steps:
• Discard sequences with N bases
• Discard sequences that come from a consensus over too few source sequences
• Discard sequences with too few unique CDR3s (CDR3s_exact column)
• Discard sequences with too few unique Js (Js_exact column)
• Discard sequences identical to one of the database sequences (if DB given)
• Discard sequences that do not match a set of known good motifs
• Discard sequences that contain a stop codon (has_stop column)
• Discard near-duplicate sequences
• Discard cross-mapping artifacts
• Discard sequences whose “allele ratio” is too low.
If a whitelist of sequences is provided (by default, this is the input V gene database),
then the candidates that appear on it
• are not checked for the cluster size criterion,
• do not need to match a set of known good motifs,
• are never considered near-duplicates (but they are checked for cross-mapping and for the
allele ratio),
• are allowed to contain a stop codon.
Whitelisting allows IgDiscover to identify known germline sequences that are expressed at
low levels in a library. If enabled with whitelist: true (the default) in the pregermline
and germline filter sections of the configuration file, the sequences present in the
starting database are treated as validated germline sequences and will not be discarded if
due to too small cluster size as long as they fulfill the remaining criteria
(unique_cdr3s, unique_js etc.).
You can see why a candidate was filtered by inspecting the annotated_V_*.tab files
Cross-mapping artifacts
If two very similar sequences appear in the database used by IgBLAST, then sequencing
errors may lead to one sequence incorrectly being assigned to the other. This is
particularly problematic if one of the sequences is highly expressed while the other is
not expressed at all. The not expressed sequence is even included in the list of V gene
candidates because it is in the input database and therefore whitelisted. We call this a
“cross-mapping artifact”.
The germline filtering step of IgDiscover therefore aims to eliminate cross-mapping
artifacts by checking all pairs of sequences for the following:
• The two sequences have a distance of 1,
• they are both in the database for that particular iteration (only then can cross-mapping
occur)
• the ratio between the expression levels of the two sequences (using the cluster_size
field in the candidates.tab file) is less than the value cross_mapping_ratio defined in
the configuration file (0.02 by default).
If all that is the case, then the sequence with the lower expression is discarded.
Allele-ratio filtering
When multiple alleles of the same gene appear in the list of V gene candidates, such as
IGHV1-2*02 and IGHV1-2*04, the germline filter computes the ratio of the values in the
exact and the clonotypes columns between them. If the ratio is under the configured
threshold, the candidate with the lower count is discarded. See the exact_ratio and
clonotype_ratio settings in the germline_filter and pregermline_filter sections of the
configuration file.
New in version 0.7.0.
Data from the Sequence Read Archive (SRA)
To work with datasets from the Sequence Read Archive, you may want to use the tool
fastq-dump, which can download the reads in the format required by IgDiscover. You just
need to know the accession number, such as “SRR2905710” and then run this command to
download the files to the current directory:
fastq-dump --split-files --gzip SRR2905710
The --split-files option ensures that the paired-end reads are stored in two separate
files, one for the forward and one for the reverse read, respectively. (If you do not
provide it, you will get an interleaved FASTQ file that currently cannot be read by
IgDiscover). The --gzip option creates compressed output. The command creates two files
in the current directory. In the above example, they would be named SRR2905710_1.fastq.gz
and SRR2905710_2.fastq.gz.
The program fastq-dump is part of the SRA toolkit. On Debian-derived Linux distributions,
you can typically install it with sudo apt-get install sra-toolkit. On Conda, install it
with conda install -c bioconda sra-tools.
Does random subsampling influence results?
Random subsampling indeed influences somewhat which sequences are found by the cluster
analysis, particularly in the beginning. However, the probability is large that all highly
expressed sequences are represented in the random sample. Also, due to the database
growing with subsequent iterations, the set of sequences assigned to a single database
gene becomes smaller and more homogeneous. This makes it increasingly likely that also
sequences expressed at lower levels result in a cluster since they now make up a larger
fraction of each subsample.
Also, many of the clusters which are captured in one subsample but not in the other are
artifacts that are then filtered out anyway by the pre-germline or germline filter.
On human data with a nearly complete starting database, the subsampling seems to have no
influence at all, as we determined experimentally. We repeated a run of the program four
times on the same human dataset, using identical parameters each time except that the
subsampling was done in a different way. Although intermediate results differed, all four
personalized databases that the program produced were exactly identical.
Concordance is lower, though, when the input database is not as complete as the human one.
The way in which random subsampling is done is modified by the seed configuration setting,
which is set to 1 by default. If its value is the same for two different runs of the
program with otherwise identical settings, the numbers chosen by the random number
generator will be the same and therefore also subsampling will be done in an identical
way. This makes runs of the program reproducible. In order to test how results differ when
subsampling is done in a different way, change the seed to a different value.
Logging the program’s output to a file
When you report a bug or unusual behavior to us, we might ask you to send us the output of
igdiscover run. You can send its output to a file by running the program like this:
igdiscover run >& logfile.txt
And here is how to send the logging output to a file and also see the output in your
terminal at the same time (but you lose the colors):
igdiscover run |& tee logfile.txt
Caching of IgBLAST results and of merged reads
Sometimes you may want to re-analyze a dataset multiple times with different filter
settings. To speed this up, IgDiscover can cache the results of two of the most
time-consuming steps, read-merging with PEAR and running IgBLAST.
The cache is disabled by default as it uses a lot of disk space. To enable the cache,
create a file named ~/.config/igdiscover.conf with the following contents:
use_cache: true
If you do so, a directory named ~/.cache/igdiscover/ is created the next time you run
IgDiscover and all IgBLAST results as well as merged reads from PEAR are stored there. On
subsequent runs, the existing result is used directly without calling the respective
program, which speeds up the pipeline considerably.
The cache is only used when we are certain that the results will indeed be the same. For
example, if the IgBLAST program version or th V/D/J database changes, the cached result is
not used.
The files in the cache are compressed, but the cache may still get large over time. You
can delete the cache with rm -r ~/.cache/igdiscover to free the space.
You should also delete the cache when updating to a newer IgBLAST version as the old
results will not be used anymore.
Terms
Analysis directory
The directory that was created with igdiscover init. Separate ones are created for
each experiment. When you used igdiscover init myexperiment, the analysis directory
would be myexperiment/.
Starting database
The initial list of V/D/J genes. These are expected to be in FASTA format and are
copied into the database/ directory within each analysis directory.
QUESTIONS AND ANSWERS
How many sequences are needed to discover germline V gene sequences?
Library sizes of several hundred thousand sequences are required for V gene discovery,
with even higher numbers necessary for full database production. For example, IgM library
sizes of 750,000 to 1,000,000 sequences for heavy chain databases and 1.5 to 2 million
sequences for light chain databases.
Can IgDiscover analyze IgG libraries?
IgDiscover has been developed to identify germline databases from libraries that contain
substantial fractions of unswitched antibody sequences. We recommend IgM libraries for
heavy chain V gene identification and IgKappa and IgLambda libraries for light chain
identification. IgDiscover can identify a proportion of gemline sequences in IgG
libraries but the process is much more efficient with IgM libraries, enabling the full set
of germline sequences to be discovered.
Can IgDiscover analyze a previously sequenced library?
Yes, IgDiscover accepts both unpaired FASTQ files and paired FASTA files but the program
should be made aware which is being used, see input requirements.
Do the positions of the PCR primers make a difference to the output?
Yes. For accurate V gene discovery, all primer sequences must be external to the V gene
sequences. For example, forward multiplex amplification primers should be present in the
leader sequence or 5' UTR, and reverse amplification primers should be located in the
constant region, preferably close to the 5' border of the constant region. Primers that
are present in framework 1 region or J segments are not recommended for library
production.
What are the advantages to 5'-RACE compared to multiplex PCR for IgDiscover analysis?
Both 5'-RACE and multiplex PCR have their own advantages.
5'-RACE will enable library production from species where the upstream V gene sequence is
unknown. The output of the upstream subcommand in IgDiscovery enables the identification
of consensus leader and 5'-UTR sequences for each of the identified germline V genes, that
can subsequenctly be used for primer design for either multiplex PCR or for monoclonal
antibody amplification sets.
Multiplex PCR is recommended for species where the upstream sequences are well
characterized. Multiplex amplification products are shorter than 5'-RACE products and
therefore will be easier to pair and will have less length associated sequence errors.
What is meant by 'starting database'?
The starting database refers to the folder that contains the three FASTA files necessary
for the process of iterative V gene discovery to begin. IgDiscover uses the standalone
IgBLAST program for comparative assignment of sequences to the starting database. Because
IgBlast requires three files (for example V.fasta, D.fasta, J.fasta), three FASTA files
should be included in the database folder for each analysis to proceed.
In the case of light chains (that do not contain D segments), a dummy D segment file
should be included as IgBLAST will not proceed if it does not see three files in the
database folder. It is sufficient to save the following sequence as a fasta file and
rename it D.fasta, for example, for it to function as the dummy D.fasta file for human
light chain analysis:
>D_ummy
GGGGGGGGGG
How can I use the IMGT database as a starting database?
Since we do not have permission to distribute IMGT database files with IgDiscover, you
need to download them directly from IMGT. See the section about obtaining a V/D/J
database.
How do I change the parameters of the program?
By editing the configuration file.
Where do I find the individualized database produced by IgDiscover?
The final germline database in FASTA format is in your analysis directory in the
subdirectory final/database/. The V.fasta file contains the new list of V genes. The
D.fasta and J.fasta files are unchanged from the starting database.
A phylogenetic tree of the V sequences can be found in final/dendrogram_V.pdf.
For more details of how that database was created, you need to inspect the files created
in the last iteration of the discovery process, located in iteration-xx, where xx is the
number of iterations configured in the igdiscover.yaml configuration file. For example, if
three iterations were used, look into iteration-03/.
Most interesting in that folder are likely
• the linkage cluster analysis plots in iteration-03/clusterplots/,
• the error histograms in iteration-03/errorhistograms.pdf, which contain the windowed
cluster analysis figures.
• Details about the individualized database in new_V_germline.tab in tab-separated-value
format
The new_V_germline.fasta file is identical to the one in final/database/V.fasta
What does the _S1234 at the end of same gene names mean?
Please see the Section on gene names.
ADVANCED TOPICS
IgDiscover itself does not (yet) come with all imaginable analysis facilities built into
it. However, it creates many files (mostly with tables) that can be used for custom
analysis. For example, all .tab files (in particular assigned.tab.gz and candidates.tab)
can be opened and inspected in a spreadsheet application such as LibreOffice. From there,
you can do basic tasks such as sorting from the menu of that application.
Often, these facilities are not enough, however, and some basic understanding of the
command-line is helpful. Clearly, this is not as convenient as working in a graphical user
interface (GUI), but we do not currently have the resources to provide one for IgDiscover.
To alleviate this somewhat, we provide here instructions for a few things that you may
want to do with the IgDiscover result files.
Extract all sequences that match any database gene exactly
The candidates.tab file tells you for each discovered sequence how often an exact match of
that sequence was found in your input reads. A high number of exact matches is a good
indication that the candidate is actually a new gene or allele. In order to find the
original reads that correspond to those matches, you can look at the filtered.tab.gz file
and extract all rows where the V_errors column is zero.
First, run this on the filtered.tab.gz file:
zcat filtered.tab.gz | head -n 1 | tr '\t' '\n' | nl
This will enumerate the columns in the file. Take a note of the index that the V_errors
column has. In newer pipeline versions, the index is 21. Then extract all rows of the file
where that field is equal to zero:
zcat filtered.tab.gz | awk -vFS="t" '$21 == 0 || NR == 1' > exact.tab
If the column wasn’t 21, then replace the $21 appropriately. The part where it says NR ==
1 ensures that the column headings are also printed.
Extra configuration settings
Some configuration settings are not documented in the default igdiscover.yaml file since
they rarely need to be changed.
# Leave empty or choose a species name supported by IgBLAST:
# human, mouse, rabbit, rat, rhesus_monkey
# This setting is not used anywhere except that it is passed
# to IgBLAST using the -organism option. Since we provide IgBLAST
# with our own gene databases, it seems this has no effect.
species:
# Which program to use for computing multiple alignments. This is used for
# computing consens sequences.
# Choose 'mafft', 'clustalo', 'muscle' or 'muscle-fast'.
# 'muscle-fast' runs muscle with parameters "-maxiters 1 -diags".
#
#multialign_program: muscle-fast
DEVELOPMENT
• Source code
• Report an issue
Installing the development version
To use the most recent IgDiscover version from Git, follow these steps.
1. If you haven’t done so, install miniconda. See the first steps of the regular
installation instructions. Do not install IgDiscover, yet!
2. Clone the IgDiscover repository:
git clone https://github.com/NBISweden/IgDiscover.git
(Use the git@ URL instead if you are a developer.)
4. Create a new Conda environment using the environment.yml file in the repository:
cd IgDiscover
conda env create -n igdiscover -f environment.yml
You can choose a different environment name by changing the name after the -n
parameter. This may be necessary, when you already have a regular (non-developer)
IgDiscover installation in an igdiscover environment that you don’t want to overwrite.
5. Activate the environment:
source activate igdiscover
(Or use whichever name you chose above.)
6. Install IgDiscover in “editable” mode:
python3 -m pip install -e .
Whenever you want to update the software:
cd IgDiscover
git pull
It may also be necessary to repeat the python3 -m pip install -e . step.
IgBLAST result cache
For development, in particular when running tests repeatedly, you should enable the
IgBLAST result cache. The cache stores IgBLAST output. If the same dataset with the same
dataset is run a second time, the result is retrieved from the cache and IgBLAST is not
re-run. This saves a lot of time when re-running datasets, but may also fill up the cache
directory ~/.cache/igdiscover/. Also, in production, datasets are usually not re-run with
the same settings, which is why caching is disabled by default.
To enable the cache, create a file ~/.config/igdiscover.conf with the following content:
use_cache: true
The file is in YAML format, but at the moment, no other settings are supported.
Building the documentation
Go to the doc/ directory in the repository, then run:
make
to build the documentation locally. Open _build/html/index.html in a browser. The layout
is different from the version shown on Read the Docs, but allows you to preview any
changes you may have made.
Making a release
We use versioneer to manage version numbers. It extracts the version number from the most
recent tag in Git. Thus, to increment the version number, create a Git tag:
git tag v0.5
The v prefix is mandatory.
Then:
• tests/run.sh
• python3 setup.py sdist
• twine upload sdist/igdiscover-0.10.tar.gz
• Update bioconda recipe
Removing IgDiscover from a Linux system
If you have been playing around with different installation methods (pip, conda, git,
python3 setup.py install etc.) you may have multiple copies of IgDiscover on your system
and you will likely run into problems on updates. Here is a list you can follow in order
to get rid of the installations as preparation for a clean re-install. Do not add sudo to
the commands below if you get permission problems, unless explicitly told to do so! If one
of the steps does not work, that is fine, just continue.
1. Delete miniconda: Run the command which conda. The output will be something like
/home/myusername/miniconda3/bin/conda. The part before bin/conda is the miniconda
installation directory. Delete that folder. In this case, you would need to delete
miniconda3 in /home/myusername.
2. Run pip3 uninstall igdiscover. If this runs successfully and prints some messages about
removing files, then repeat the same command! Do this until you get a message telling
you that the package cannot be uninstalled because it is not installed.
3. Repeat the previous step, but with pip3 uninstall sqt.
4. If you have a directory named .local within your home directory, you may want to rename
it: mv .local dot-local-backup You can also delete it, but there is a small risk that
other software (not IgDiscover) uses that directory. The directory is hidden, so a
normal ls will not show it. Use ls -la while in your home directory to see it.
5. If you have ever used sudo to install IgDiscover, you may have an installation in
/usr/local/. You can try to remove it with sudo pip3 uninstall igdiscover.
6. Delete the cloned Git repository if you have one. This is the directory in which you
run git pull.
Finally, you can follow the normal installation instructions and then the developer
installation instructions.
CHANGES
v0.11 (2018-11-27)
• The IgBLAST cache is now disabled by default. We assume that, in most cases, datasets
will not be re-run with the exact same parameters, and then it only fills up the disk.
Delete your cache with rm -r ~/.cache/igdiscover to reclaim the space. To enable the
cache, create a file ~/.config/igdiscover.conf with the contents use_cache: true.
• If you choose to enable the cache, results from the PEAR merging step will now also be
cached. See also the caching documentation.
• Added detection of chimeras to the (pre-)germline filters. Any novel allele that can be
explained as a chimera of two unmodified reference alleles is marked in the
new_V_germline.tab file. This is a bit sensitive, so the candidate is currently not
discarded.
• Two additional files annotated_V_germline.tab and annotated_V_pregermline.tab are
created in each iteration during the germline filtering step. These are identical to the
candidates.tab file, except that they contain a why_filtered column that describes why a
sequence was filtered. See the documentation for this feature.
• A more realistic test dataset (v0.5), now based on human instead of rhesus data, was
prepared. The testing instructions have been updated accordingly.
• J discovery has been tuned to give fewer truncated sequences.
• Statistics are written to stats/stats.json.
• V SHM distribution plots are created automatically and written written to
v-shm-distributions.pdf in each iteration folder.
• An igdiscover dbdiff subcommand was added that can compare two FASTA files.
v0.10 (2018-05-11)
• When computing a consensus sequence, allow some sequences to be truncated in the 3' end.
Many of the discovered novel V alleles were truncated by one nucleotide in the 3' end
because IgBLAST does not always extend the alignment to the end of the V sequence. If
these slightly too short V sequences were in the majority, their consensus would lead to
a truncated sequence as well. The new consensus algorithm allows for this effect at the
3' end and can therefore more often than previously find the full sequence. Example:
TACTGTGCGAGAGA (seq 1)
TACTGTGCGAGAGA (seq 2)
TACTGTGCGAGAG- (seq 3)
TACTGTGCGAG--- (seq 4)
TACTGTGCGAG--- (seq 5)
TACTGTGCGAGAG (previous consensus)
TACTGTGCGAGAGA (new consensus)
• Add a column database_changes to the new_V_germline.tab file that describes how the
novel sequence differs from the database sequence. Example: 93C>T; 114A>G
• Allow filtering by CDR3_shared_ratio and do so by default (needs documentation)
• Cache the edit distance when computing the distance matrix. Speeds up the discover
command slightly.
• discover: Use more than six CPU cores if available
• igblast: Print progress every minute
v0.9 (2018-03-22)
• Implemented allele ratio filtering for J gene discovery
• J genes are discovered as part of the pipeline (previously, one needed to run the
discoverj script manually)
• In each iteration, dendrograms are now created not only for V genes, but also for D and
J genes. The file names are dendrogram_D.pdf, dendrogram_J.pdf
• The V dendrograms are now in dendrogram_V.pdf (no longer V_dendrogram.pdf). This puts
all the dendrograms together when looking at the files in the iteration directory.
• The V_usage.tab and V_usage.pdf files are no longer created. Instead, expressed_V.tab
and expressed_V.pdf are created. These contain similar information, but an allele-ratio
filter is used to filter out artifacts.
• Similarly, expressed_D.tab and expressed_J.tab and their .pdf counterparts are created
in each iteration.
• Removed parse subcommand (functionality is in the igblast subcommand)
• New CDR3 detection method (only heavy chain sequences): CDR3 start/end coordinates are
pre-computed using the database V and J sequences. Increases detection rate to 99%
(previously less than 90%).
• Remove the ability to check discovered genes for required motifs. This has never worked
well.
• Add a column clonotypes to the candidates.tab that tries to count how many clonotypes
are associated with a single candidate (using only exact occurrences). This is intended
to replace the CDR3s_exact column.
• Add an exact_ratio to the germline filtering options. This checks the ratio between the
exact V occurrence counts (exact column) between alleles.
• Germline filtering option allele_ratio was renamed to clonotypes_ratio
• Implement a cache for IgBLAST results. When the same dataset is re-analyzed, possibly
with different parameters, the cached results are used instead of re-running IgBLAST,
which saves a lot of time. If the V/D/J database or the IgBLAST version has changed,
results are not re-used.
v0.8.0 (2017-06-20)
• Add a barcodes_exact column to the candidates table. It gives the number of unique
barcode sequences that were used by the sequences in the set of exact sequences. Also,
add a configuration setting barcode_consensus that can turn off consensus taking of
barcode groups, which needs to be set to false for barcodes_exact to work.
• Add a Ds_exact column to candidates table.
• Add a D_coverage configuration option.
• The pre-processing filtering step no longer reads in the full table of IgBLAST
assignments, but filters the table piece by piece. Memory usage for this step therefore
does not depend anymore on the dataset size and should always be below 1 GB.
• The functionality of the parse subcommand has been integrated into the igblast
subcommand. This means that igdiscover igblast now directly outputs a result table
(assigned.tab). This makes it easier to use that subcommand directly instead of only via
the workflow.
• The igblast subcommand now always runs makeblastdb by itself and deletes the BLAST
database afterwards. This reduces clutter and ensures the database is always up to date.
• Remove the library_name configuration setting. Instead, the library_name is now always
the same as the name of analysis directory.
v0.7.0 (2017-05-04)
• Add an “allele ratio” criterion to the germline filter to further reduce the number of
false positives. The filter is activated by default and can be configured through the
allele_ratio setting in the configuration file. See the documentation for how it works.
• Ignore the CDR3-encoding bases whenever comparing two V gene sequences.
• Avoid finding 5'-truncated V genes by extending found hits towards the 5' end.
• By default, candidate sequences are no longer merged if they are nearly identical. That
is, the differences setting within the two germline filter configuration sections is now
set to zero by default. Previously, we believed the merging would remove some false
positives, but it turns out we also miss true positives. It also seems that with the
other changes in this version we also no longer get the particular false positives the
setting was supposed to catch.
• Implement an experimental discoverj script for J gene discovery. It is curently not run
automatically as part of igdiscover run. See igdiscover discoverj --help for how to run
it manually.
• Add a config subcommand, which can be used to change the configuration file from the
command-line.
• Add a V_CDR3_start column to the assigned.tab/filtered.tab tables. It describes where
the CDR3 starts within the V sequence.
• Similarly, add a CDR3_start column to the new_V_germline.tab file describing where the
CDR3 starts within a discovered V sequence. It is computed by using the most common
CDR3 start of the sequences within the cluster.
• Rename the compose subcommand to germlinefilter.
• The init subcommand automatically fixes certain problems in the input database
(duplicate sequences, empty records, duplicate sequence names). Previously, it would
complain, but the user would have to fix the problems themselves.
• Move source code to GitHub
• Set up automatic code testing (continuous integration) via Travis
• Many documentation improvements
v0.6.0 (2016-12-07)
• The FASTA files of the input V/D/J gene lists now need to be named V.fasta, D.fasta and
J.fasta. The species name is no longer part of the file name. This should reduce
confusion when working with species not supported by IgBLAST.
• The species: configuration setting in the configuration can (and should) now be left
empty. Its only use was that it is passed to IgBLAST, but since IgDiscover provides
IgBLAST with its own V/D/J sequences anyway, it does not seem to make a difference.
• A “cross-mapping” detection has been added, which should reduce the number of false
positives. See the documentation for an explanation.
• Novel sequences identical to a database sequence no longer get the _S1234 suffix.
• No longer trim trim the initial G run in sequences (due to RACE) by default. It is now a
configuration setting.
• Add cdr3_location configuration setting: It allows to set whether to use a CDR3 in
addition to the barcode for grouping sequences.
• Create a groups.tab.gz file by default (describing the de-barcoded groups)
• The pre-processing filter is now configurable. See the preprocessing_filter section in
the configuration file.
• Many improvements to the documentation
• Extended and fixed unit tests. These are now run via a CI system.
• Statistics in JSON format are written to stats/stats.json.
• IgBLAST 1.5.0 output can now be parsed. Parsing is also faster by 25%.
• More helpful warning message when no sequences were discovered in an iteration.
• Drop support for Python 3.3.
v0.5 (2016-09-01)
• V sequences of the input database are now whitelisted by default. The meaning of the
whitelist configuration option has changed: If set to false, those sequences are no
longer whitelisted. To whitelist additional sequences, create a whitelist.fasta file as
before.
• Sequences with stop codons are now filtered out by default.
• Use more stringent germline filtering parameters by default.
v0.4 (2016-08-24)
• It is now possible to install and run IgDiscover on OS X. Appropriate Conda packages are
available on bioconda.
• Add column has_stop to candidates.tab, which indicates whether the candidate sequence
contains a stop codon.
• Add a configuration option that makes it possible to disable the 5' motif check by
setting check_motifs: false (the looks_like_V column is ignored in this case).
• Make it possible to whitelist known sequences: If a found gene candidate appears in that
list, the sequence is included in the list of discovered sequences even when it would
otherwise not pass filtering criteria. To enable this, just add a whitelist.fasta file
to the project directory before starting the analysis.
• The criteria for germline filter and pre-germline filter are now configurable: See
germline_filter and pre_germline_filter sections in the configuration file.
• Different runs of IgDiscover with the same parameters on the same input files will now
give the same results. See the seed parameter in the configuration, also on how to get
non-reproducible results as before.
• Both the germline and pre-germline filter are now applied in each iteration. Instead of
the new_V_database.fasta file, two files named new_V_germline.fasta and
new_V_pregermline.fasta are created.
• The compose subcommand now outputs a filtered version of the candidates.tab file in
addition to a FASTA file. The table contains columns closest_whitelist, which is the
name of the closest whitelist sequence, and whitelist_diff, which is the number of
differences to that whitelist sequence.
v0.3 (2016-08-08)
• Optionally, sequences are not renamed in the assigned.tab file, but retain their
original name as in the FASTA or FASTQ file. Set rename: false in the configuration file
to get this behavior.
• Started an “advanced” section in the manual.
v0.2
• IgDiscover can now also detect kappa and lambda light chain V genes (VK, VL)
AUTHOR
Marcel Martin
COPYRIGHT
2015-2023, Marcel Martin