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)
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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
0.11 Mar 10, 2023 IGDISCOVER(1)