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Sequencing of individual barcoded cDNAs using Pacific Biosciences and Oxford Nanopore Technologies reveals platform-specific error patterns.

TitleSequencing of individual barcoded cDNAs using Pacific Biosciences and Oxford Nanopore Technologies reveals platform-specific error patterns.
Publication TypeJournal Article
Year of Publication2022
AuthorsMikheenko A, Prjibelski AD, Joglekar A, Tilgner HU
JournalGenome Res
Volume32
Issue4
Pagination726-737
Date Published2022 Apr
ISSN1549-5469
KeywordsDNA, Complementary, High-Throughput Nucleotide Sequencing, Nanopores, RNA, Sequence Analysis, DNA, Technology
Abstract

Long-read transcriptomics require understanding error sources inherent to technologies. Current approaches cannot compare methods for an individual RNA molecule. Here, we present a novel platform-comparison method that combines barcoding strategies and long-read sequencing to sequence cDNA copies representing an individual RNA molecule on both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We compare these long-read pairs in terms of sequence content and isoform patterns. Although individual read pairs show high similarity, we find differences in (1) aligned length, (2) transcription start site (TSS), (3) polyadenylation site (poly(A)-site) assignment, and (4) exon-intron structures. Overall, 25% of read pairs disagree on either TSS, poly(A)-site, or splice site. Intron-chain disagreement typically arises from alignment errors of microexons and complicated splice sites. Our single-molecule technology comparison reveals that inconsistencies are often caused by sequencing error-induced inaccurate ONT alignments, especially to downstream GUNNGU donor motifs. However, annotation-disagreeing upstream shifts in NAGNAG acceptors in ONT are often confirmed by PacBio and are thus likely real. In both barcoded and nonbarcoded ONT reads, we find that intron number and proximity of GU/AGs better predict inconsistencies with the annotation than read quality alone. We summarize these findings in an annotation-based algorithm for spliced alignment correction that improves subsequent transcript construction with ONT reads.

DOI10.1101/gr.276405.121
Alternate JournalGenome Res
PubMed ID35301264