articleNature MethodsMar 13, 2025HYBRID OA

A systematic benchmark of Nanopore long-read RNA sequencing for transcript-level analysis in human cell lines

YCYing ChenNDN. DavidsonYKYuk Kei WanFYFei YaoYSYan Su

Agency for Science, Technology and Research · Genome Institute of Singapore · +19 more institutions

PubMed
Indexed incrossrefpubmed

Abstract

Abstract The human genome contains instructions to transcribe more than 200,000 RNAs. However, many RNA transcripts are generated from the same gene, resulting in alternative isoforms that are highly similar and that remain difficult to quantify. To evaluate the ability to study RNA transcript expression, we profiled seven human cell lines with five different RNA-sequencing protocols, including short-read cDNA, Nanopore long-read direct RNA, amplification-free direct cDNA and PCR-amplified cDNA sequencing, and PacBio IsoSeq, with multiple spike-in controls, and additional transcriptome-wide N 6 -methyladenosine profiling data. We describe differences in read length, coverage, throughput and transcript…

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Topics & keywords

Topics
Keywords
  • RNA
  • Complementary DNA
  • Biology
  • Nanopore sequencing
  • Computational biology
  • Gene isoform
  • RNA-Seq
  • Transcriptome
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