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Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing

View ORCID ProfileSøren M. Karst, View ORCID ProfileRyan M. Ziels, View ORCID ProfileRasmus H. Kirkegaard, View ORCID ProfileMads Albertsen
doi: https://doi.org/10.1101/645903
Søren M. Karst
1Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Denmark.
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Ryan M. Ziels
2Department of Civil Engineering, The University of British Columbia, Vancouver, Canada
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Rasmus H. Kirkegaard
1Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Denmark.
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Mads Albertsen
1Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Denmark.
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  • For correspondence: ma@bio.aau.dk
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Abstract

High-throughput amplicon sequencing of large genomic regions represents a challenge for existing short-read technologies. Long-read technologies can in theory sequence large genomic regions, but they currently suffer from high error rates. Here, we report a high-throughput amplicon sequencing approach that combines unique molecular identifiers (UMIs) with Oxford Nanopore sequencing to generate single-molecule consensus sequences of large genomic regions. We demonstrate the approach by generating nearly 10,000 full-length ribosomal RNA (rRNA) operons of roughly 4,400 bp in length from a mock microbial community consisting of eight bacterial species using a single Oxford Nanopore MinION flowcell. The mean error rate of the consensus sequences was 0.03%, with no detectable chimeras due to a rigorous UMI-barcode filtering strategy. The simplicity and accessibility of this method paves way for widespread use of high-accuracy amplicon sequencing in a variety of genomic applications.

Footnotes

  • - included new and updated references. - added missing link to raw data. - fixed typos. - improved figure text.

  • 1 Oligonucleotide sequences © 2007-2018 Illumina, Inc. All rights reserved. Illumina adaptor sequences were used as synthetic priming sites, as they are proven to work robustly for PCR amplification and to allow the option to validate libraries with Illumina sequencing, which is useful during troubleshooting.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted May 28, 2019.
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Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing
Søren M. Karst, Ryan M. Ziels, Rasmus H. Kirkegaard, Mads Albertsen
bioRxiv 645903; doi: https://doi.org/10.1101/645903
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Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing
Søren M. Karst, Ryan M. Ziels, Rasmus H. Kirkegaard, Mads Albertsen
bioRxiv 645903; doi: https://doi.org/10.1101/645903

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