README.md

bulkBCRseq : isotyper

Bulk BCR-seq processing package used in Fitzpatrick et al., Nature (2020). The original (legacy) package/scripts was provided by Dr. Rachael Bashford-Rogers (Oxford).

This repository is a python3 reimplementation of the original python2 scripts (found in legacy branch); the original script is an older version of what seems to be now at https://github.com/rbr1/BCR_TCR_PROCESSING_PIPELINE.

Requires python>=3.8 (or python==2.7.9 if using the legacy branch).

Citation

Please cite the following papers:

Fitzpatrick, Z., Frazer, G., Ferro, A., Clare, S., Bouladoux, N., Ferdinand, J., Tuong, Z.K., Negro-Demontel, M.L., Kumar, N., Suchanek, O. and Tajsic, T., 2020. Gut-educated IgA plasma cells defend the meningeal venous sinuses. Nature, 587(7834), pp.472-476.

Bashford-Rogers, R.J., Palser, A.L., Huntly, B.J., Rance, R., Vassiliou, G.S., Follows, G.A. and Kellam, P., 2013. Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations. Genome research, 23(11), pp.1874-1884.

Bashford-Rogers, R.J.M., Bergamaschi, L., McKinney, E.F., Pombal, D.C., Mescia, F., Lee, J.C., Thomas, D.C., Flint, S.M., Kellam, P., Jayne, D.R.W. and Lyons, P.A., 2019. Analysis of the B cell receptor repertoire in six immune-mediated diseases. Nature, 574(7776), pp.122-126.

Setup:

# create a conda virtual environment
# sample for python 3 set up, switch to python 2 where appropriate
# install miniconda
# see https://docs.conda.io/en/latest/miniconda.html#linux-installers
wget https://repo.anaconda.com/miniconda/Miniconda3-py39_4.12.0-Linux-x86_64.sh
bash Miniconda3-py39_4.12.0-Linux-x86_64.sh
eval "$(/path/to/miniconda2/bin/conda shell.bash hook)"
conda init
conda create --name isotyper python=3.9

# clone this repository
git clone https://github.com/clatworthylab/bulkBCRseq

# change into the directory and install dependencies
cd bulkBCRseq
conda env update --name isotyper --file environment.yml

# either run this everytime or just
# export to your ~/.bashrc or ~/.bash_profile
export PYTHONPATH=/path/to/bulkBCRseq:$PYTHONPATH
export REF_PATH=/lustre/scratch117/core/sciops_repository/cram_cache/%2s/%2s/%s:/lustre/scratch118/core/sciops_repository/cram_cache/%2s/%2s/%s:URL=http:://refcache.dnapipelines.sanger.ac.uk::8000/%s

# always activate the environment before proceeding
conda activate isotyper
# main usage
python /path/to/bulkBCRseq/isotyper.py [options]

usage: isotyper.py [-h] [-i INPUT] [-s STEP] [-l LENGTH] [-dr] [-b] [-c CORES] [-m MEM] [-q QUEUE] [-p PROJECT] [-g GROUP]

options:
  -h, --help            show this help message and exit

main arguments:
  -i INPUT, --input INPUT
                        input meta.txt file to run isotyper.
                        file must contain the following four columns:
                            1st column - name of sample.
                            2nd column - path to input file. Either .cram file or read 1 fastq(.gz) file.
                            3rd column - path to output folder.
                            4th column - organism. Either HOMO_SAPIENS or MUS_MUSCULUS.
                            no column names allowed.
  -s STEP, --step STEP  step to perform:
                            1 - Convert raw sequencing files to fastq and perform QC.
                            2 - Trim and filter reads.
                            3 - Generate networks.
                            4 - Generate network statistics.
  -l LENGTH, --length LENGTH
                        minimum length of reads to keep. [Default 100]
  -dr, --dryrun         if passed, prints commands but don't actually run.

bsub arguments:
  -b, --bsub            if passed, submits each row in meta.txt file as a job to bsub.
  -c CORES, --cores CORES
                        number of cores to run this on. [Default 10]
  -m MEM, --mem MEM     job memory request. [Default 8000]
  -q QUEUE, --queue QUEUE
                        job queue to submit to. [Default normal]
  -p PROJECT, --project PROJECT
                        sanger project to send as job. [Default team205]
  -g GROUP, --group GROUP
                        sanger group to send as job. [Default teichlab]

Note!

If you are starting from fastq files directly, please change the 2nd column in the .txt file (path to .cram) to path to _R1_001.fastq.gz (read1) instead. If your read1/read2 suffix isn't this pattern, please modify the R1PATTERN and R2PATTERN variables file after cloning this repository, in the _settings.py directly:

bulkBCRseq/isotyper/utilities/_settings.py

Lines 25 to 27 in 5d310de

	# change here if necessary
	R1PATTERN = "_R1_001"
	R2PATTERN = "_R2_001"

this also means that your files should be named with the suffix like:

<sample1>_R1_001.fastq.gz
<sample1>_R2_001.fastq.gz

Basic usage

# initial QC
python isotyper.py -i meta.txt -s 1
# trimming
python isotyper.py -i meta.txt -s 2
# generate network
python isotyper.py -i meta.txt -s 3
# generate network statistic
python isotyper.py -i meta.txt -s 4

If using Sanger's farm:

# initial QC
python isotyper.py -i meta.txt -s 1 --bsub
# trimming
python isotyper.py -i meta.txt -s 2 --bsub
# generate network
python isotyper.py -i meta.txt -s 3 --bsub
# generate network statistic
python isotyper.py -i meta.txt -s 4 --bsub

Take a look here for example files to provide to the tool.

Post-processing

After running steps 1 to 4, please annotate the Fully_reduced_{sample_id}.fasta file for downstream analysis. You can annotate with IMGT/HighV-QUEST or via other software e.g. MiXCR in shotgun mode.

mixcr analyze shotgun -s hsa --starting-material rna --receptor-type igh Fully_reduced_{sample_id}.fasta {sample_id} 
# export to AIRR format
mixcr exportAirr --imgt-gaps in.[vdjca|clns|clna] out.tsv

To generate the network plots, you would use the node table (Att_{sample_id}.txt) and edge table (Edges_{sample_id}.txt) and feed it into a graphing software e.g. networkx/igraph and continue as per normal. The orphan folder has example scripts (probably buggy) on how to use python-igraph to generate the plots.