Real-Time Targeted Exome Enrichment in Single-cell Long-read Sequencing

More than 90% of multi-exonic human genes undergo Alternative Splicing (AS), a process that gives rise to distinct RNA isoforms from a single gene. Characterizing cell-type specific isoform expression improves the granularity of transcriptomic signatures and cellular functions. However, in the cDNA library, many pre-mRNA molecules may not have been spliced. Therefore, enriching for exonic reads can increase the statistical power and reveal cell type specific AS patterns. This allows for the understanding of splicing induced cellular functions with a finer resolution.To facilitate the study of AS using long-read sequencing data, we have previously developed an exome probe approach to enrich for spliced reads of target genes. However, the three-day procedure is time-consuming and expensive. Read Until API allows for real-time enrichment of genetic regions during the sequencing process by ejecting non-target reads. We utilized tools to enrich for exonic reads of target genes of interest without the caveats of our previous probe-based approach. In parallel with sequencing, open-source software Readfish performs rapid identification of targeted sequences and selectively rejects untargeted reads in real time. Here we demonstrate real-time targeted exon enrichment to increase the abundance of exonic reads being sequenced without additional time or cost.We performed real-time enrichment by targeting spliced transcripts from 3,377 human genes implicated in brain functions and related diseases. Optimization was performed to maximize enrichment for our single-cell long-read RNA samples. Our results revealed a notable up to 1.82x increase in the total number of spliced on-target reads (from 259k to 470k reads), and a 1.80x improvement in on-target rates compared to control (from 34.01% to 95.38%). Surprisingly, a significant 1.92~3.36X increase of spliced on-target reads can be observed in targeting the genes with low abundance reads among all 3,377 genes. This technique serves as a more cost and time-effective alternative for the exome probe-based enrichment method.We demonstrated that real-time exon enrichment is an effective, affordable, flexible, and time-saving approach to enrich for exonic sequences of target genes from the whole single cell cDNA population, enabling more statistical power of downstream computational analysis compared to the exome probe enrichment. In the future, this technology could potentially be applied to more datasets to conduct interesting investigations, such as disease-healthy comparison and new isoform identification.