Pre-processed IgH repertoire sequencing data from BioProject PRJNA748239

Data Processing

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>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:

 

C_CALL                    Isotype subclass

SEQUENCE_ID                Sequence identifier

V_CALL                    V segment gene and allele

D_CALL                    D segment gene and allele

J_CALL                    J segment gene and allele

JUNCTION_LENGTH            Junction length

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

DUPCOUNT                    UMI count for the given unique sequence

ISOTYPE                    Constant region primer (isotype)

MU_COUNT_CDR_R            Number of replacement mutations in CDR region

MU_COUNT_CDR_S            Number of silent mutations in CDR region

MU_COUNT_FWR_R            Number of replacement mutations in FWR region

MU_COUNT_FWR_S            Number of silent mutations in FWR region

MUT_TOTAL                    Total number of mutations in V gene 

NP_LENGTH                    Total number of N and P additions            

SEQUENCE_INPUT            Full length sequence

SEQUENCE_IMGT                Gapped IMGT sequence

V_GERM_START_VDJ            position of the first nucleotide in ungapped V germline sequence alignment

JUNCTION                    Junction nucleotide sequence

GERMLINE_IMGT_D_MASK        IMGT-gapped germline nucleotide sequence with ns masking the NP1-D-NP2 regions

CDR3_AA_GRAVY       CDR3 hydrophobicity

CDR3_AA_BULK       CDR3 bulkiness

CDR3_AA_ALIPHATIC        Normalized aliphatic index

CDR3_AA_POLARITY        CDR3 polarity

CDR3_AA_CHARGE        normalised net charge

CDR3_AA_BASIC        Basic side chain residue content

CDR3_AA_ACIDIC        Acidic side chain residue content

CDR3_AA_AROMATIC        aromatic side chain conten

Subset                    Defined B cell subset 

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

R_SCDR                    R/S ratio in CDR region

R_SFWR                    R/S ratio in FWR region

V_GENE                    V segment gene

D_GENE                    D segment gene

J_GENE                    J segment gene

V_FAM                    V family gene

Run                        ID of sequencing run

Sex                        Sex of the Subject

Age                        Age of the subject

UNIQUE_ID                    Subject identifier 

SAMPLE                    Sample identifier, linking back to raw data

Bcellno                    Number of input B cells

Cells                    Cell type              

 

References

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. Bioinformatics30: 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. Bioinformatics31: 3356–3358.

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

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