Removing Chimeras From Antibody Repertoire Sequencing

Revealing repertoire dynamics and obtaining antibodies of interest with sequence-and nucleotide-level resolutions via antibody repertoire sequencing(Rep-seq)have been widely adopted and fruitful in adaptive immunology studies.However,necessary polymerase chain reaction(PCR)introduces extensive chimeras leading to false discovery of antibodies and incorrect somatic hypermutations(SHMs),and thus severely misleads downstream investigations.While analyze the downloaded data from published paper,we found more public clones in intra-project donor pairs,the relationship of these intra-project donors pair was not the same with inter-project's.and the mutation rate,singleton fraction in these intra-project public clones were higher than inter-project public clones,We first analyzed the fact which may affect the formation of chimera,and we found annealing temperature,PCR cycle,amplification strategy and the similarity of the sequences can be the influence factor.With knowing the factor,we designed DUMPChimera which labels each sample with dual barcodes and each molecule with dual unique molecular identifiers(UMIs)via minimal PCR amplification to remove inter-and intra-sample chimeras and acquire accurate repertoires.In our ultra-deep testing data,DUMPChimera removed inter-sample chimeras causing unreal shared clones and constituting?15%of the reads in the library,and got rid of intra-sample chimeras with erroneous SHMs and occupying-20%of the reads.After removing these chimeras,the public clones between samples decreased,and the mutation number in the reads became fewer.By correcting the amplification biases in the PCR,the reproducibility of the library preparation became higher.Removal of these artifacts and high reproducibility of the repertoire are invaluable for the research in the field and this method sheds new light on promoting the applications of other high-throughput sequencing involving PCR in library preparation.