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A novel ultra high-throughput 16S rRNA amplicon sequencing library preparation method on the Illumina HiSeq platform

Eric J. de Muinck, Pål Trosvik, Gregor D. Gilfillan, Arvind Y. M. Sundaram
doi: https://doi.org/10.1101/124057
Eric J. de Muinck
1Centre for Ecological and Evolutionary Synthesis, Dept. of Biosciences, University of Oslo, Oslo, Norway
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Pål Trosvik
1Centre for Ecological and Evolutionary Synthesis, Dept. of Biosciences, University of Oslo, Oslo, Norway
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Gregor D. Gilfillan
2Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
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Arvind Y. M. Sundaram
2Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway
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  • For correspondence: arvind.sundaram@medisin.uio.no
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Abstract

Background Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost, and benchmarking the techniques so that potential sources of bias can be better characterized.

Results We present a triple-index amplicon sequencing strategy that uses a two-stage PCR protocol. The strategy was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.

Conclusions Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

Footnotes

  • Author full name and email address: EJM: ericdemuinck{at}gmail.com, PT: pal.trosvik{at}ibv.uio.no, GDG: gregorg{at}medisin.uio.no, AYMS: arvind.sundaram{at}medisin.uio.no

  • List of abbreviations

    rRNA
    Ribosomal RNA
    PCR
    Polymerase chain reaction
    OTU
    Operational taxonomic unit
    BLAST
    Basic local alignment search tool
    MDS
    Multidimensional scaling
    PBS
    Phosphate buffered saline
    ANOSIM
    Analysis of similarity
    GC content
    Guanine-cytosine content
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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    Posted April 05, 2017.
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    A novel ultra high-throughput 16S rRNA amplicon sequencing library preparation method on the Illumina HiSeq platform
    Eric J. de Muinck, Pål Trosvik, Gregor D. Gilfillan, Arvind Y. M. Sundaram
    bioRxiv 124057; doi: https://doi.org/10.1101/124057
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    A novel ultra high-throughput 16S rRNA amplicon sequencing library preparation method on the Illumina HiSeq platform
    Eric J. de Muinck, Pål Trosvik, Gregor D. Gilfillan, Arvind Y. M. Sundaram
    bioRxiv 124057; doi: https://doi.org/10.1101/124057

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