{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T00:09:50Z","timestamp":1792195790831,"version":"4.4.0"},"reference-count":26,"publisher":"National Academy of Sciences","issue":"39","license":[{"start":{"date-parts":[[2018,9,10]],"date-time":"2018-09-10T00:00:00Z","timestamp":1536537600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010336","name":"Hellman Foundation","doi-asserted-by":"publisher","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}],"id":[{"id":"10.13039\/100010336","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000051","name":"HHS | NIH | National Human Genome Research Institute","doi-asserted-by":"publisher","award":["1T32HG008345-01"],"award-info":[{"award-number":["1T32HG008345-01"]}],"id":[{"id":"10.13039\/100000051","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2018,9,25]]},"abstract":"<jats:p>High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples.<\/jats:p>","DOI":"10.1073\/pnas.1806447115","type":"journal-article","created":{"date-parts":[[2018,9,10]],"date-time":"2018-09-10T15:05:20Z","timestamp":1536591920000},"page":"9726-9731","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":254,"title":["Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA"],"prefix":"10.1073","volume":"115","author":[{"given":"Roger","family":"Volden","sequence":"first","affiliation":[{"name":"Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064;"},{"name":"Genomics Institute, University of California, Santa Cruz, CA 95064;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Theron","family":"Palmer","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064;"},{"name":"Genomics Institute, University of California, Santa Cruz, CA 95064;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashley","family":"Byrne","sequence":"additional","affiliation":[{"name":"Genomics Institute, University of California, Santa Cruz, CA 95064;"},{"name":"Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Cruz, CA 95064;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charles","family":"Cole","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064;"},{"name":"Genomics Institute, University of California, Santa Cruz, CA 95064;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert J.","family":"Schmitz","sequence":"additional","affiliation":[{"name":"Department of Genetics, University of Georgia, Athens, GA 30602"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard E.","family":"Green","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064;"},{"name":"Genomics Institute, University of California, Santa Cruz, CA 95064;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Vollmers","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064;"},{"name":"Genomics Institute, University of California, Santa Cruz, CA 95064;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2018,9,10]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/nmeth.1226","article-title":"Mapping and quantifying mammalian transcriptomes by RNA-seq","volume":"5","author":"Mortazavi A","year":"2008","unstructured":"A Mortazavi, BA Williams, K McCue, L Schaeffer, B Wold, Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods 5, 621\u2013628 (2008).","journal-title":"Nat Methods"},{"key":"e_1_3_4_2_2","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1038\/nbt.3242","article-title":"Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events","volume":"33","author":"Tilgner H","year":"2015","unstructured":"H Tilgner, , Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events. Nat Biotechnol 33, 736\u2013742 (2015).","journal-title":"Nat Biotechnol"},{"key":"e_1_3_4_3_2","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1101\/gr.230516.117","article-title":"Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome","volume":"28","author":"Tilgner H","year":"2017","unstructured":"H Tilgner, , Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome. Genome Res 28, 231\u2013242 (2017).","journal-title":"Genome Res"},{"key":"e_1_3_4_4_2","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1038\/nbt.2705","article-title":"A single-molecule long-read survey of the human transcriptome","volume":"31","author":"Sharon D","year":"2013","unstructured":"D Sharon, H Tilgner, F Grubert, M Snyder, A single-molecule long-read survey of the human transcriptome. Nat Biotechnol 31, 1009\u20131014 (2013).","journal-title":"Nat Biotechnol"},{"key":"e_1_3_4_5_2","doi-asserted-by":"crossref","first-page":"12065","DOI":"10.1038\/ncomms12065","article-title":"Long-read sequencing and de novo assembly of a Chinese genome","volume":"7","author":"Shi L","year":"2016","unstructured":"L Shi, , Long-read sequencing and de novo assembly of a Chinese genome. Nat Commun 7, 12065 (2016).","journal-title":"Nat Commun"},{"key":"e_1_3_4_6_2","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1186\/s12864-017-3691-9","article-title":"Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human","volume":"18","author":"Kuo RI","year":"2017","unstructured":"RI Kuo, , Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human. BMC Genomics 18, 323 (2017).","journal-title":"BMC Genomics"},{"key":"e_1_3_4_7_2","doi-asserted-by":"crossref","first-page":"16027","DOI":"10.1038\/ncomms16027","article-title":"Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells","volume":"8","author":"Byrne A","year":"2017","unstructured":"A Byrne, , Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. 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