title: Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods.
abstract: BACKGROUND
  Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers.
  
  
  RESULTS
  Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5' Rapid amplification of cDNA ends (5'RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA.
  
  
  CONCLUSIONS
  Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.
authors.forename: Rachael J M
authors.initials: RJ
authors.lastname: Bashford-Rogers
authors.forename: Anne L
authors.initials: AL
authors.lastname: Palser
authors.forename: Saad F
authors.initials: SF
authors.lastname: Idris
authors.forename: Lisa
authors.initials: L
authors.lastname: Carter
authors.forename: Michael
authors.initials: M
authors.lastname: Epstein
authors.forename: Robin E
authors.initials: RE
authors.lastname: Callard
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Douek
authors.forename: George S
authors.initials: GS
authors.lastname: Vassiliou
authors.forename: George A
authors.initials: GA
authors.lastname: Follows
authors.forename: Mike
authors.initials: M
authors.lastname: Hubank
authors.forename: Paul
authors.initials: P
authors.lastname: Kellam
mesh_terms: D004247:DNA / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D012313:RNA / Q000235:genetics; D011947:Receptors, Antigen, B-Cell / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013269:Stochastic Processes
chemical_list: D011947:Receptors, Antigen, B-Cell; D012313:RNA; D004247:DNA