Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

Abstract

The sequence upstream of antibody variable region (Antibody Upstream Sequence, or AUS) consists of 5’ untranslated region (5’ UTR) and two leader regions, L-PART1 and L-PART2. The sequence variations in AUS affect the efficiency of PCR amplification, mRNA translation, and subsequent PCR-based antibody quantification as well as antibody engineering. Despite their importance, the diversity of AUSs has long been neglected. Utilizing the rapid amplification of cDNA ends (5’RACE) and high-throughput antibody repertoire sequencing (Rep-Seq) technique, we acquired full-length AUSs for human, rhesus macaque (RM), cynomolgus macaque (CM), mouse, and rat. We designed a bioinformatics pipeline and discovered 2,957 unique AUSs, corresponding to 2,786 and 1,159 unique sequences for 5’ UTR and leader, respectively. Comparing with the leader records in the international ImMunoGeneTics (IMGT), while 529 were identical, 313 were with single nucleotide polymorphisms (SNPs), 280 were totally new, and 37 updated the incomplete records. The diversity of AUSs’ impact on related antibody biology was also probed. Taken together, our findings would facilitate Rep-Seq primer design for capturing antibodies comprehensively and efficiently as well as provide a valuable resource for antibody engineering and the studies of antibody at the molecular level.

Poorly Expressed Alleles of Several Human Immunoglobulin Heavy Chain Variable Genes are Common in the Human Population

“…An analysis of the upstream region (part of the 5’UTR and the sequence encoding the leader sequence) of the current data was possible as the NGS-derived sequence information had been derived from libraries generated by 5’-RACE technology ( 16 ). This comparison could thus be carried out both using genomic and transcriptome-based, inferred ( 24 , 25 ) sequence information. The intron sequence found within the signal peptide-encoding sequence, and the sequence upstream of the 5’-end of the mRNA had to be compared to genomic sequence information found in public repositories ( Supplementary Figure 10 ).…”

“…The observed 5’UTR-leader sequences of the transcriptomes derived from IGHV4-4*01 of the present investigation did not exactly match neither the corresponding sequence of the allele or of highly expressed alleles IGHV4-4*02 or IGHV4-4*07, as recoded in the IMGT database. However, gene inference studies have identified such alternative 5’UTR-leader sequences in IGHV4-4*02 in a number of subjects ( 25 ; Huang et al manuscript in preparation). Furthermore, the leader sequence intron of genomic sequences of IGHV4-4*01 and IGHV4-4*02 are identical.…”

“…These sequences were compared to relevant genomic annotations as described in the IMGT database and by some additional genomic sequences found in GenBank. These sequences were also compared to those defined in a separate study ( 24 ) studying the data sets also investigated in the present study, and to the most prevalent 5’UTR-leader sequence variants reported in a study of unrelated data sets ( 25 ).…”

“…The IMGT/LIGM-DB reference sequences is indicated as primary while other sequences derived from the IMGT website are indicated as secondary. Inferred sequences were obtained from publications by Zhu et al ( 25 ) (of which only the more common variants are shown) and Mikocziova et al ( 24 ). The number after these entries indicate the number of cases in which precisely this sequence had been inferred in these studies.…”

Computational Inference, Validation, and Analysis of 5’UTR-Leader Sequences of Alleles of Immunoglobulin Heavy Chain Variable Genes

“…Bioinformatic tools developed for studies of large transcriptomic repertoire data sets, such as IgDiscover ( 9 ) and IMGT/HighV-QUEST ( 17 ), are already able to capture parts of the 5’UTRs and the signal peptide-encoding part of the genes in many existing NGS data sets. Recent studies, however, have suggested that the diversities of 5’UTR and leader sequences are not well represented in the IMGT database ( 14 , 18 ), strongly arguing that such information ought to be updated to enable analysis of the role of these regions in gene expression and functionality. Heterozygosity in 5’UTR and leader sequences may also be used in sequence haplotyping efforts to assess gene expression from individual chromosomes ( 19 , 20 ) even in cases when their associated IGHV genes are identical, thereby allowing further development of our understanding of these genes in a broader context.…”