Abstract
Long-read sequencing holds great potential for transcriptome analysis because it offers researchers an affordable method to annotate the transcriptomes of non-model organisms. This, in turn, will greatly benefit future work on less-researched organisms like unicellular eukaryotes that cannot rely on large consortia to generate these transcriptome annotations. However, to realize this potential, several remaining molecular and computational challenges will have to be overcome. In this review, we have outlined the limitations of short-read sequencing technology and how long-read sequencing technology overcomes these limitations. We have also highlighted the unique challenges still present for long-read sequencing technology and provided some suggestions on how to overcome these challenges going forward.This article is part of a discussion meeting issue ‘Single cell ecology’.
Highlights
The rapid progress and application of sequencing technology after the completion of the Human Genome Project has led to a vastly expanded knowledge of the genome sequences present in the eukaryotic tree of life
While it remains to be determined what accuracy will be sufficient for reliably identifying regular transcript isoforms, increasing the accuracy of individual reads to beyond 99% will be required for single cell complementary DNA (cDNA) demultiplexing and the analysis of individual transcripts that contain unique sequences not encoded in the genome, i.e. B and T cell receptor transcripts, as well as transcripts that contain base modifications
There is little doubt in our minds that full-length transcriptome sequencing using long-read technologies is the future of transcriptome annotation because it has too many inherent advantages over short-read approaches
Summary
The rapid progress and application of sequencing technology after the completion of the Human Genome Project has led to a vastly expanded knowledge of the genome sequences present in the eukaryotic tree of life. Sequencing technology is reaching a point where it might soon be feasible to affordably generate high-quality genome references and transcriptome annotations of a much wider range of organisms than previously possible— including unicellular eukaryotes. Genome assembly is entering a golden age where high-quality ‘centromere-to-telomere’ genome sequences can be assembled through a mix of several technologies, including short-read sequencing, linked short-read sequencing (e.g. Hi-C), long-read sequencing and optical mapping [2,3,4] These powerful and relatively affordable approaches are going to be of outsize benefit for non-model organisms from unicellular eukaryotes to polar bears that in the past did not receive the attention and large sums of money required to generate a high-quality genome royalsocietypublishing.org/journal/rstb Phil. We discuss the potential and limitations of long-read based full-length transcriptome sequencing for transcriptome annotation and lay out a path towards realizing this potential
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