Abstract
BackgroundAlternative mRNA isoform usage is an important source of protein diversity in mammalian cells. This phenomenon has been extensively studied in bulk tissues, however, it remains unclear how this diversity is reflected in single cells.ResultsHere we use long-read sequencing technology combined with unique molecular identifiers (UMIs) to reveal patterns of alternative full-length isoform expression in single cells from the mouse brain. We found a surprising amount of isoform diversity, even after applying a conservative definition of what constitutes an isoform. Genes tend to have one or a few isoforms highly expressed and a larger number of isoforms expressed at a low level. However, for many genes, nearly every sequenced mRNA molecule was unique, and many events affected coding regions suggesting previously unknown protein diversity in single cells. Exon junctions in coding regions were less prone to splicing errors than those in non-coding regions, indicating purifying selection on splice donor and acceptor efficiency.ConclusionsOur findings indicate that mRNA isoform diversity is an important source of biological variability also in single cells.
Highlights
Alternative mRNA isoform usage is an important source of protein diversity in mammalian cells
The cDNA was previously prepared using Single-cell tagged reverse transcription (STRT)/C1 [23], which resulted in fulllength cDNA normally sequenced from the 5′ end, to indicate only the transcription start site
We instead sequenced each cDNA sample using Pacific Biosciences Single Molecule Real Time (PacBio SMRT) technology [24], which generated long reads often comprising the entire length of each cDNA molecule
Summary
Alternative mRNA isoform usage is an important source of protein diversity in mammalian cells. This phenomenon has been extensively studied in bulk tissues, it remains unclear how this diversity is reflected in single cells. Alternative mRNA isoform usage is prevalent in mammalian genomes, and allows the creation of a highly diverse set of proteins from a relatively small number of genes. Alternative isoform usage has been studied at the 5′ end [5]; for exon splicing [3] and at the 3′ end [6]. The most common approach to study alternative isoform usage is by second generation sequencing, but
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