DOI: 10.5281/zenodo.5155564
title: Pre-processed IgH repertoire sequencing data from BioProject PRJNA748239

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) software_versions pRESTO:0.5.3,Change-O:0.3.4,IgBlast 1.6.1, stampy1.0.21. shazam0.1.8 quality_thresholds FilterSeq.py pRESTO Q&gt;20 paired_reads_assembly AssemblePairs.py pRESTO minlen 10 maxerror 0.3 alpha 0.0001 primer_match_cutoffs MaskPrimers.py pRESTO C primer & V primer maxerror 0.2 consensus_building BuildConsensus.py pRESTO maxerror 0.1 maxgap 0.5 collapsing_method CollapseSeq.py pRESTO germline_database IMGT Format 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>NP_LENGTH</strong> Total number of N and P additions<strong> </strong> <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>CDR3_AA_GRAVY</strong> CDR3 hydrophobicity <strong>CDR3_AA_BULK</strong> CDR3 bulkiness <strong>CDR3_AA_ALIPHATIC</strong> Normalized aliphatic index <strong>CDR3_AA_POLARITY</strong> CDR3 polarity <strong>CDR3_AA_CHARGE</strong> normalised net charge <strong>CDR3_AA_BASIC</strong> Basic side chain residue content <strong>CDR3_AA_ACIDIC</strong> Acidic side chain residue content <strong>CDR3_AA_AROMATIC</strong> aromatic side chain conten <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_GENE</strong> V segment gene <strong>D_GENE</strong> D segment gene <strong>J_GENE</strong> J segment gene <strong>V_FAM</strong> V family gene <strong>Run</strong> ID of sequencing run <strong>Sex</strong> Sex of the Subject <strong>Age</strong> Age of the subject <strong>UNIQUE_ID</strong> Subject identifier <strong>SAMPLE</strong> Sample identifier, linking back to raw data <strong>Bcellno</strong> Number of input B cells <strong>Cells</strong> Cell type <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.