Single-cell sequencing has allowed the non-averaged measurements of genotypic and phenotypic differences between individual cells on a genome-wide scale. More recently, simultaneous measurements of genotypic and expression heterogeneity were demonstrated with a number of single-cell whole genome and transcriptome paired sequencing (scWGS/RNA-seq) methods. However, these methods suffer from inherent bias and limitations. One of them is their inability to capture the full transcriptome but only mRNA, in order to avoid the cost of sequencing the abundant ribosomal RNA (rRNA) that are not of interest. To address this challenge, we have developed a CRISPR-based method named scDASH (single-cell depletion of abundant sequences by hybridisation) to deplete the rRNA, and demonstrated a 3.5-fold...[ Read more ]

Single-cell sequencing has allowed the non-averaged measurements of genotypic and phenotypic differences between individual cells on a genome-wide scale. More recently, simultaneous measurements of genotypic and expression heterogeneity were demonstrated with a number of single-cell whole genome and transcriptome paired sequencing (scWGS/RNA-seq) methods. However, these methods suffer from inherent bias and limitations. One of them is their inability to capture the full transcriptome but only mRNA, in order to avoid the cost of sequencing the abundant ribosomal RNA (rRNA) that are not of interest. To address this challenge, we have developed a CRISPR-based method named scDASH (single-cell depletion of abundant sequences by hybridisation) to deplete the rRNA, and demonstrated a 3.5-fold enrichment of the rest of the transcriptome including both polyadenylated mRNA and non-polyadenylated transcripts. Another bias of current scWGS/RNA-seq methods originates from having to physically segregate nuclear genomic DNA from cytoplasmic mRNA. While this approach could avoid potential cross-contamination of DNA and RNA reads, it comes at the expense of higher dropout rates at both allelic and transcript levels. As a solution, our group recently developed a novel scWGS/RNA-seq method that adopts a one-tube reaction format. We will discuss one of the strategies we attempted to avoid DNA/RNA cross-labelling during the development of our method.