Introduction

Vgene is an R package for analyzing bulk targeted DNA sequencing data of B-cell receptors (BCR) generated from the Invivoscribe LymphoTrack IGH FR1 assay kit. It processes the sequencing data end-to-end, from raw FASTQ files to annotated clone clusters and lineage trees.

Installation of the package 

# install.packages("remotes")
remotes::install_gitlab(
  "nfdi4immuno/tools/v-gene",
  host = "https://codebase.helmholtz.cloud"
)

External tools to be installed by the user

The following command-line tools must be installed and on your PATH before running the pipeline:

Tool How to verify
NCBI BLAST+ blastn -version
GNU parallel parallel --version
PHYLIP dnapars dnapars

Input files for the package

1) Samples file

A CSV file named samples.csv in the working directory with one row per sample. Three columns are required:

number,name,folder
1,Patient_77_PB-CSW,Patient_77
2,Patient_77_PB-MD27,Patient_77
3,Patient_78_PB-CSW,Patient_78
Column Type Description Example
number Integer Sample index 1
name Character Unique sample identifier Patient_77_PB-CSW
folder Character Donor/group label Patient_77

2) FASTQ files

Paired-end FASTQ files (*.fastq.gz) should all reside in a single directory. run_pipeline() creates an R1_and_R2/ folder inside the working directory and copies them there automatically.

Running the Pipeline

library(vgene)

run_pipeline(
  workdir       = "v-gene",
  samples_file  = "samples.csv",
  fastq_dir     = "R1_and_R2",
  ighv_path     = system.file("extdata", "makeblastdb",
                               "IGHV_reference.fasta", package = "vgene"),
  blast_dir     = "ncbi-blast-2.16.0/bin",
  phylip_exec   = "/opt/phylip-3.697/exe/dnapars"
)

Output folder structure

v-gene/
└── Patient_77/                            # Per-patient output
    ├── output_clones.tsv   # annotated vgene clones with AIRR fields
    └── lineage_plots/                     # one plot per clone
        ├── clone_1.pdf
        ├── clone_2.pdf