Leveraging Advances In Long-Read Sequencing to Improve Analysis of Host Immune Responses 2332674

Abstract

Abstract Introduction Short-read based technologies have dominated high-throughput sequencing workflows for years. However, many complex sequences such as immune genes exhibit repetitive regions, duplications, or splicing variations and it is challenging to assemble or align them accurately using short reads. Since long-read sequencing has traditionally been lower-throughput and higher in cost, recent advances conferred by Pacbio’s Kinnex platform may help address these limitations, but new protocol developments are needed to enable the use of existing material prepared for standard short-read platforms. Methods To establish new Kinnex based workflows that are compatible with commonly used single cell platforms, we processed cDNAs generated from various rhesus tissue types using the 10x Genomics technology according to the MAS-ISO-seq protocol through TSO artifact removal and then enriched IG, TCR, or MHC transcripts using custom designed primers. Separately, existing Qiaseq based bulk human TCR libraries, containing an Illumina Nextera backbone, were amplified using construct specific primers. In both cases, we introduced compatibility to the PacBio Kinnex 16S rRNA workflow through custom primers? and incorporated amplicons into the Kinnex workflow at the point of array PCR. Resulting libraries were sequenced on PacBio’s Revio platform. Results Successful gene-targeted single cell library constructions were accomplished for the 10x Genomics cDNAs, and existing Qiagen bulk TCR libraries were converted to SMRTbell libraries. Increased mapping efficiencies were observed across the entire transcripts, particularly concerning full-length VDJ transcripts. Conclusion Adaptation of existing library materials into a Kinnex-based protocol allows for streamlining of multiple constructs into a single workflow while expanding accessibility to PacBio’s long-read sequencing platform, resulting in a more affordable and productive approach for improving data quality and facilitating the study of immune system expression. Funding Source NIH U19 AI131471, NIH R01 AI175411, CHHE grant # ES025128 Topic Categories Technological Innovations in Immunology (TECH)