NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform

Calus, S. T., Ijaz, U. Z. and Pinto, A. (2018) NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform. GigaScience, 7(12), giy140. (doi:10.1093/gigascience/giy140) (PMID:30476081) (PMCID:PMC6298384)

Calus, S. T., Ijaz, U. Z. and Pinto, A. (2018) NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform. GigaScience, 7(12), giy140. (doi:10.1093/gigascience/giy140) (PMID:30476081) (PMCID:PMC6298384)

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Abstract

Background: Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output. Results: In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences. Conclusions: NanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Pinto, Dr Ameet and Calus, Szymon and Ijaz, Dr Umer Zeeshan
Authors: Calus, S. T., Ijaz, U. Z., and Pinto, A.
College/School:College of Science and Engineering > School of Engineering
College of Science and Engineering > School of Engineering > Infrastructure and Environment
Journal Name:GigaScience
Publisher:Oxford University Press
ISSN:2047-217X
ISSN (Online):2047-217X
Published Online:23 November 2018
Copyright Holders:Copyright © 2018 The Authors
First Published:First published in GigaScience 7(12):giy140
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
665801Healthy drinking waterAmeet PintoEngineering and Physical Sciences Research Council (EPSRC)EP/M016811/1ENG - ENGINEERING INFRASTRUCTURE & ENVIR
652772Understanding microbial community through in situ environmental 'omic data synthesisUmer Zeeshan IjazNatural Environment Research Council (NERC)NE/L011956/1ENG - ENGINEERING INFRASTRUCTURE & ENVIR