DOI: 10.5281/zenodo.2640393
title: Pre-processed B cell receptor repertoire sequencing data from BioProject PRJNA527941

description: <strong>Data Processing</strong>

 

Samples were demultiplexed via their Illumina indices, and processed using the Immcantation toolkit(1,2). Raw fastq files were filtered based on a quality score threshold of 20. Paired reads were joined if they had a minimum length of 10 nt, maximum error rate of 0.3 and a significance threshold of 0.0001. Reads with identical UMI were collapsed to a consensus sequence. Reads with identical full-length sequence and identical constant primer but differing UMI were further collapsed. Sequences were then submitted to IgBlast (3) for VDJ assignment and sequence annotation. Constant region sequences were mapped to germline using Stampy(4). The number and type of V gene mutations was calculated using the shazam R package.(2)

 

<strong>software_versions</strong>            pRESTO:0.5.3,Change-O:0.3.4,IgBlast 1.6.1, stampy1.0.21. shazam0.1.8

<strong>quality_thresholds</strong>            FilterSeq.py pRESTO Q&gt;20

<strong>paired_reads_assembly</strong>        AssemblePairs.py pRESTO minlen 10 maxerror 0.3 alpha 0.0001

<strong>primer_match_cutoffs</strong>        MaskPrimers.py pRESTO C primer & V primer maxerror 0.2

<strong>consensus_building</strong>        BuildConsensus.py pRESTO maxerror 0.1 maxgap 0.5

<strong>collapsing_method</strong>        CollapseSeq.py pRESTO

<strong>germline_database        </strong>IMGT

 

<strong>Format</strong>

 

Processed sequences are provided in a tab delimited file format, including the following annotations:

 

<strong>C_CALL                    </strong>Isotype subclass

<strong>SEQUENCE_ID                </strong>Sequence identifier

<strong>V_CALL                    </strong>V segment gene and allele

<strong>D_CALL                    </strong>D segment gene and allele

<strong>J_CALL                    </strong>J segment gene and allele

<strong>JUNCTION_LENGTH            </strong>Junction length

<strong>CONSCOUNT                </strong>Raw read count from which UMI consensus sequences were generated, summed over all UMIs for the given unique sequence.

<strong>DUPCOUNT                    </strong>UMI count for the given unique sequence

<strong>ISOTYPE                    </strong>Constant region primer (isotype)

<strong>MU_COUNT_CDR_R            </strong>Number of replacement mutations in CDR region

<strong>MU_COUNT_CDR_S            </strong>Number of silent mutations in CDR region

<strong>MU_COUNT_FWR_R            </strong>Number of replacement mutations in FWR region

<strong>MU_COUNT_FWR_S            </strong>Number of silent mutations in FWR region

<strong>MUT_TOTAL                    </strong>Total number of mutations in V gene 

<strong>SEQUENCE_INPUT            </strong>Full length sequence

<strong>SEQUENCE_IMGT                </strong>Gapped IMGT sequence

<strong>V_GERM_START_VDJ            </strong>position of the first nucleotide in ungapped V germline sequence alignment

<strong>JUNCTION                    </strong>Junction nucleotide sequence

<strong>GERMLINE_IMGT_D_MASK        </strong>IMGT-gapped germline nucleotide sequence with ns masking the NP1-D-NP2 regions

<strong>Run                        </strong>ID of sequencing run

<strong>Sample_type                </strong>The tissue sampled (e.g Peripheral Blood, bone marrow, ..)

<strong>Sex                        </strong>Sex of the Subject

<strong>Age                        </strong>Age of the subject

<strong>UNIQUE_ID                    </strong>Subject identifier 

<strong>SAMPLE_ID                    </strong>Sample identifier, linking back to raw data

<strong>Subset                    </strong>Defined B cell subset 

<strong>Repertoire                    </strong>Defined B cell repertoire (Naive, Memory IgM/IgD, IgA, IgG)

<strong>R_SCDR                    </strong>R/S ratio in CDR region

<strong>R_SFWR                    </strong>R/S ratio in FWR region

<strong>V_FAM                    </strong>V family gene

<strong>V_GENE                    </strong>V segment gene

<strong>D_GENE                    </strong>D segment gene

<strong>J_GENE                    </strong>J segment gene

<strong>Clust_Rank                    </strong>Cluster rank

<strong>Clust_REPRES                </strong>Cluster representative

<strong>Clust_SIZE                    </strong>Cluster size

<strong>Clust_MAXFREQ                </strong>Cluster maximum frequency

<strong>Clust_SHAREDNESS            </strong>Cluster sharedness

<strong>CDR3_AA_GRAVY                </strong>CDR3 hydrophobicity index

<strong>CDR3_AA_CHARGE            </strong>CDR3 charge

<strong>CDRH3PDB                    </strong>CDRH3 PDB (Structure) code

<strong>H1Canon                    </strong>H1 Canonical class

<strong>H2Canon                    </strong>H2 Canonical class

<strong>H1_GERMLINE                </strong>H1 Germline Canonical class

<strong>H2_GERMLINE                </strong>H2 Germline Canonical class

 

<strong>References</strong>

1. Vander Heiden, J. A., G. Yaari, M. Uduman, J. N. H. Stern, K. C. O’Connor, D. A. Hafler, F. Vigneault, and S. H. Kleinstein. 2014. PRESTO: A toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires. <em>Bioinformatics</em>30: 1930–1932.

2. Gupta, N. T., J. A. Vander Heiden, M. Uduman, D. Gadala-Maria, G. Yaari, and S. H. Kleinstein. 2015. Change-O: A toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data. <em>Bioinformatics</em>31: 3356–3358.

3. Ye, J., N. Ma, T. L. Madden, and J. M. Ostell. 2013. IgBLAST: an immunoglobulin variable domain sequence analysis tool. <em>Nucleic Acids Res.</em>41.

4. Lunter, G., and M. Goodson. 2011. Stampy: A statistical algorithm for sensitive and fast mapping of Illumina sequence reads. <em>Genome Res.</em>21: 936–939.