High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

被引：230

作者：

Karst, Soren M. ^{[1
]}

Ziels, Ryan M. ^{[2
]}

Kirkegaard, Rasmus H. ^{[1
]}

Sorensen, Emil A. ^{[1
]}

McDonald, Daniel ^{[3
]}

Zhu, Qiyun ^{[3
]}

Knight, Rob ^{[3
,4
,5
,6
]}

Albertsen, Mads ^{[1
]}

机构：

[1] Aalborg Univ, Dept Chem & Biosci, Ctr Microbial Commun, Aalborg, Denmark

[2] Univ British Columbia, Dept Civil Engn, Vancouver, BC, Canada

[3] Univ Calif San Diego, Dept Pediat, La Jolla, CA 92093 USA

[4] Univ Calif San Diego, Dept Bioengn, La Jolla, CA 92093 USA

[5] Univ Calif San Diego, Ctr Microbiome Innovat, La Jolla, CA 92093 USA

[6] Univ Calif San Diego, Dept Comp Sci & Engn, La Jolla, CA 92093 USA

来源：

NATURE METHODS | 2021年 / 18卷 / 02期

基金：

加拿大自然科学与工程研究理事会;

关键词：

PCR PRIMERS; SEARCH; GENES;

D O I：

10.1038/s41592-020-01041-y

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 (similar to 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 15x (ONT R10.3), 25x (ONT R9.4.1) and 3x (Pacific Biosciences circular consensus sequencing) is needed, which provides a mean error rate of 0.0042%, 0.0041% and 0.0007%, respectively.

引用

页码：165 / +

页数：11

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