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. 2022;2453:345–377. doi: 10.1007/978-1-0716-2115-8_19

Bulk Sequencing from mRNA with UMI for Evaluation of B-Cell Isotype and Clonal Evolution: A Method by the AIRR Community.

Nidhi Gupta, Susanna Marquez, Cinque Soto, Elaine C Chen, Magnolia L Bostick, Ulrik Stervbo, Andrew Farmer
PMCID: PMC9761522  PMID: 35622335

Abstract

During the course of an immune response to a virus such as influenza, B cells undergo activation, clonal expansion, isotype switching, and somatic hypermutation (SHM). Members of an antigen-experienced B-cell clone can have different sequence features including SHM in the immunoglobulin heavy-chain V (IGHV) gene and can use the same IGVH gene in combination with different constant regions or isotypes (e.g., IgM, IgG, IgA). To study these features of expanded clones in an immune response by AIRR-seq, we provide a bulk RNA-based sequencing experimental procedure with unique molecular identifiers (UMIs) and the accompanying bioinformatics analytical workflow.

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References

  1. Vander Heiden JA, Yaari G, Uduman M, Stern JNH, O’Connor KC, Hafler DA et al (2014) pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires. Bioinformatics 30:1930–1932. https://doi.org/10.1093/bioinformatics/btu138 doi: 10.1093/bioinformatics/btu138. [DOI] [PMC free article] [PubMed]
  2. Mukherjee S, Huntemann M, Ivanova N, Kyrpides NC, Pati A (2015) Large-scale contamination of microbial isolate genomes by Illumina PhiX control. Stand Genomic Sci 10:18. https://doi.org/10.1186/1944-3277-10-18 doi: 10.1186/1944-3277-10-18. [DOI] [PMC free article] [PubMed]
  3. Giudicelli V, Chaume D, Lefranc M-P (2005) IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Res 33:D256–D261. https://doi.org/10.1093/nar/gki010 doi: 10.1093/nar/gki010. [DOI] [PMC free article] [PubMed]
  4. Ye J, Ma N, Madden TL, Ostell JM (2013) IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res 41:W34–W40. https://doi.org/10.1093/nar/gkt382 doi: 10.1093/nar/gkt382. [DOI] [PMC free article] [PubMed]
  5. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340 doi: 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed]
  6. Gupta NT, Vander Heiden JA, Uduman M, Gadala-Maria D, Yaari G, Kleinstein SH (2015) Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data. Bioinformatics 31:3356–3358. https://doi.org/10.1093/bioinformatics/btv359 doi: 10.1093/bioinformatics/btv359. [DOI] [PMC free article] [PubMed]
  7. Hoehn KB, Pybus OG, Kleinstein SH (2020) Phylogenetic analysis of migration, differentiation, and class switching in B cells. Immunology. https://doi.org/10.1101/2020.05.30.124446 doi: 10.1101/2020.05.30.124446. [DOI] [PMC free article] [PubMed]

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