Dual UMIs and Dual Barcodes With Minimal PCR Amplification Removes Artifacts and Acquires Accurate Antibody Repertoire

Abstract

Antibody repertoire sequencing (Rep-seq) has been widely used to reveal repertoire dynamics and to interrogate antibodies of interest at single nucleotide-level resolution. However, polymerase chain reaction (PCR) amplification introduces extensive artifacts including chimeras and nucleotide errors, leading to false discovery of antibodies and incorrect assessment of somatic hypermutations (SHMs) which subsequently mislead downstream investigations. Here, a novel approach named DUMPArts, which improves the accuracy of antibody repertoires by labeling each sample with dual barcodes and each molecule with dual unique molecular identifiers (UMIs) via minimal PCR amplification to remove artifacts, is developed. Tested by ultra-deep Rep-seq data, DUMPArts removed inter-sample chimeras, which cause artifactual shared clones and constitute approximately 15% of reads in the library, as well as intra-sample chimeras with erroneous SHMs and constituting approximately 20% of the reads, and corrected base errors and amplification biases by consensus building. The removal of these artifacts will provide an accurate assessment of antibody repertoires and benefit related studies, especially mAb discovery and antibody-guided vaccine design.

Multi-tissue architecture of the adaptive immune receptor repertoire in the cynomolgus macaque

“…The two main sources of errors in typical HTS-based approaches are sample preparation (reverse transcription and PCR) and errors inherent to the DNA sequencing platform 10,44 . Our DUMPArts method eliminates the intrinsic artifacts in AIRR pro ling 13 . In this study, we further modi ed the protocol, attaching the Illumina sequencing adapter to the amplicons during the PCR process to enable direct sequencing -LibFree DUMPArts (Fig.…”

“…1D). We previously showed that using dual UMIs can e ciently remove the intra-sample chimeras in IgM repertoires 13 . Here, we checked the antibody groups generated by single and dual UMI.…”

“…The consensus was dropped if it obtained ambiguous nucleotides, and the remaining consensus sequences was aligned to the reference germline genes downloaded from KIMDB (http://kimdb.gkhlab.se./datasets/) and IMGT (http://www.imgt.org). Then the consensus reads were used to assemble clones by Change-O 24 The comparison of SHM rate between before and after correction by dual UMIs was described previously 13 . The information of samples was provided in Supplementary Table 1.…”

“…Because of the vital role of the AIRR in health and disease, a clearer understanding of the dynamics of the AIRR may elucidate immune status, the constituent BCRs or TCRs targeting a particular antigen, and improve therapies. With powerful new methods 13,14,15,16,17 , tools 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33 and compared in Zhang et al, Smakaj et al and Yang et al 34,35,36 , databases 37,38,39 , and platforms 40,41 , AIRR pro ling has led to great progress in many areas of immunology research, such as autoimmune diseases 42,43 , microbial infection 44 , vaccination 45 , allergy 46 , aging 47 , and cancer immunotherapy 48 . This progress has led to the identi cation of neutralizing antibodies of potential therapeutic value targeting malicious viruses such as HIV-1 49 , Ebola 50 , and In uenza 51 .…”

“…Previously, we developed DUMPArts, a novel Rep-seq approach that uses dual UMIs and dual barcodes to remove sequencing artifacts 13 . Here, we expanded on DUMPArts to directly anneal the Illumina sequencing adaptors to the amplicon during the PCR process.…”