Table 1.

Challenges and solutions to sequencing the functional antibody repertoire

Challenge	Details of challenge	Solution
Analyse the functional repertoire	Identify clinical phenotype Analyse B cells participating in an immune response of interest Perform integrated analysis of coexpressed functional genes	Focus analysis on individuals with immune phenotypes of interest Focus analysis on functional B cell populations, such as plasmablasts, antigen-specific memory B cells, plasma cells or tissue-infiltrating B cells Cell barcode to enable analysis of coexpressed functional genes in B cell subsets
Overcome the scale necessary for comprehensive repertoire analysis	Analyse thousands of B cells per experiment	Use high-throughput approaches
Demonstrate function	Accurately pair IgH + IgL expressed by individual B cells Sequence full-length variable regions Have error-free sequences (correction of both RT-PCR and sequencing errors)	Use cell barcoding and linkage PCR methods Sequence entire variable regions Error correct, using cell barcodes to enable correction for RT-PCR and sequencing errors, and molecule barcodes to enable correction for sequencing but not RT-PCR errors
Overcome poor fidelity and quality	V-gene primers fail to sequence some antibodies PCR bias distorts clonal proportions RT-PCR and sequencing errors yield artefact clonal families PCR contamination produces artefacts	Use template switching or other non-V-gene primer approach to add 5′ barcode Use cell barcodes Use cell barcodes to enable correction of both types of error Use cell or molecule barcodes

Abbreviations: IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction.