Skip to main content
  • ASM Journals
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Latest Articles
    • Special Issues
    • COVID-19 Special Collection
    • Editor's Picks
    • Special Series: Sponsored Minireviews and Video Abstracts
    • Archive
  • Topics
    • Applied and Environmental Science
    • Ecological and Evolutionary Science
    • Host-Microbe Biology
    • Molecular Biology and Physiology
    • Novel Systems Biology Techniques
    • Early-Career Systems Microbiology Perspectives
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics
  • About the Journal
    • About mSystems
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • ASM Journals
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
mSystems
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Latest Articles
    • Special Issues
    • COVID-19 Special Collection
    • Editor's Picks
    • Special Series: Sponsored Minireviews and Video Abstracts
    • Archive
  • Topics
    • Applied and Environmental Science
    • Ecological and Evolutionary Science
    • Host-Microbe Biology
    • Molecular Biology and Physiology
    • Novel Systems Biology Techniques
    • Early-Career Systems Microbiology Perspectives
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics
  • About the Journal
    • About mSystems
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
Methods and Protocols | Novel Systems Biology Techniques

High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

Jeremiah J. Minich, Greg Humphrey, Rodolfo A. S. Benitez, Jon Sanders, Austin Swafford, Eric E. Allen, Rob Knight
Morgan G. I. Langille, Editor
Jeremiah J. Minich
aMarine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Greg Humphrey
bDepartment of Pediatrics, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rodolfo A. S. Benitez
bDepartment of Pediatrics, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jon Sanders
bDepartment of Pediatrics, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Austin Swafford
cCenter for Microbiome Innovation, Jacobs School of Engineering, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eric E. Allen
aMarine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, USA
cCenter for Microbiome Innovation, Jacobs School of Engineering, University of California San Diego, La Jolla, California, USA
dDivision of Biological Sciences, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rob Knight
bDepartment of Pediatrics, University of California San Diego, La Jolla, California, USA
cCenter for Microbiome Innovation, Jacobs School of Engineering, University of California San Diego, La Jolla, California, USA
eDepartment of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Morgan G. I. Langille
Dalhousie University
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/mSystems.00166-18
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements.

IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.

  • Copyright © 2018 Minich et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

View Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
Jeremiah J. Minich, Greg Humphrey, Rodolfo A. S. Benitez, Jon Sanders, Austin Swafford, Eric E. Allen, Rob Knight
mSystems Nov 2018, 3 (6) e00166-18; DOI: 10.1128/mSystems.00166-18

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print
Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this mSystems article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
(Your Name) has forwarded a page to you from mSystems
(Your Name) thought you would be interested in this article in mSystems.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
Jeremiah J. Minich, Greg Humphrey, Rodolfo A. S. Benitez, Jon Sanders, Austin Swafford, Eric E. Allen, Rob Knight
mSystems Nov 2018, 3 (6) e00166-18; DOI: 10.1128/mSystems.00166-18
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

DNA metabarcoding
Illumina MiSeq
NGS
acoustic liquid handler
automation
library preparation
metabarcoding
microbial ecology
microbiome

Related Articles

Cited By...

About

  • About mSystems
  • Author Videos
  • Board of Editors
  • Policies
  • Overleaf Pilot
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Author Warranty
  • Types of Articles
  • Getting Started
  • Ethics
  • Contact Us

Follow #mSystemsJ

@ASMicrobiology

       

 

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Online ISSN: 2379-5077