Adaptation of MAS-Seq/PacBio and ONT long read technology to the ResolveOME™ multiomic workflow for single-cell transcript isoform and DNA amplicon interrogation

Victor J. Weigman, Martin A. Newman, Isai Salas-Gonzalez, Jon S. Zawistowski

BioSkryb Genomics, Durham, NC, USA

The ability to detect accurately detect genomic structural variation (SV) is a crucial spoke in deciphering the multifaceted mechanisms of oncogenesis occurring in concert. There is often ambiguity in calling SV with short-read technologies, at both DNA and RNA levels of resolution, which can begin to be mitigated by long-read sequencing. Ascertainment of differential transcript isoform utilization (DTU) in disease or drug-resistant states also stands to directly benefit from long read and alleviate short read ambiguity. Here we embarked on studies to contrast SV and DTU calling between short and long read sequencing, at the single-cell level, with ResolveDNA genomic amplification and ResolveOME joint genomic/transcriptomic profiling [1]. We employed two long-read platforms, PacBio Revio and Oxford Nanopore Technologies (ONT) PromethION to report DNA- or RNA-level single-cell data using a cell line panel of diverse types with diverse states of copy number alteration. Transcriptomically, we devised a MAS-Seq strategy with a 16-plex of matched parental and drug-resistant triple-negative breast cancer or acute myeloid leukemia single cells as well as euploid HG001 cells to compare the full-length ResolveOME cDNA directly sequenced by the long-read platforms or fragmented for short-read readouts. Genomically, we enriched for long ResolveDNA or ResolveOME amplification products and either enzymatically fragmented these for short-read sequencing or directly sequenced using both long-read platforms. Dependent on the researcher’s biological interest, we demonstrate here the feasibility of a forked ResolveDNA/OME workflow that presents a longread option.