High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing
High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies (ONT) or Pacific Biosciences circular consensus...
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| Veröffentlicht in: | Nature methods 2021-02, Vol.18 (2), p.165-169 |
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| creator | Karst, Søren M. Ziels, Ryan M. Kirkegaard, Rasmus H. Sørensen, Emil A. McDonald, Daniel Zhu, Qiyun Knight, Rob Albertsen, Mads |
| description | High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies (ONT) or Pacific Biosciences circular consensus sequencing, yielding high-accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence ribosomal RNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities in which we observed a chimera rate |
| doi_str_mv | 10.1038/s41592-020-01041-y |
| format | Article |
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This work presents a sequencing strategy based on unique molecular identifiers that improves long-read consensus sequence accuracy of targeted amplicons as well as shotgun whole-genome fragments.</description><subject>631/337</subject><subject>631/61</subject><subject>631/61/514/1948</subject><subject>Accuracy</subject><subject>Bioinformatics</subject><subject>Biological Microscopy</subject><subject>Biological Techniques</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Conserved sequence</subject><subject>DNA sequencing</subject><subject>Genomes</subject><subject>Genomics</subject><subject>High-Throughput Nucleotide Sequencing - methods</subject><subject>Life Sciences</subject><subject>Methods</subject><subject>Microbial activity</subject><subject>Microbiota</subject><subject>Microorganisms</subject><subject>Nanopores</subject><subject>Nucleotide sequence</subject><subject>Nucleotide 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Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies (ONT) or Pacific Biosciences circular consensus sequencing, yielding high-accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence ribosomal RNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities in which we observed a chimera rate <0.02%. To reach a mean UMI consensus error rate <0.01%, a UMI read coverage of 15× (ONT R10.3), 25× (ONT R9.4.1) and 3× (Pacific Biosciences circular consensus sequencing) is needed, which provides a mean error rate of 0.0042%, 0.0041% and 0.0007%, respectively.
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| subjects | 631/337 631/61 631/61/514/1948 Accuracy Bioinformatics Biological Microscopy Biological Techniques Biomedical and Life Sciences Biomedical Engineering/Biotechnology Conserved sequence DNA sequencing Genomes Genomics High-Throughput Nucleotide Sequencing - methods Life Sciences Methods Microbial activity Microbiota Microorganisms Nanopores Nucleotide sequence Nucleotide sequencing Porosity Proteomics rRNA Workflow |
| title | High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing |
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