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Title: Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community

Journal Article · · BMC Genomics
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Background: The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composi tion and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for large initial DNA template quantities during library preparation. Results: Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. Conclusions: This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1618543
Alternate ID(s):
OSTI ID: 1378604
Journal Information:
BMC Genomics, Journal Name: BMC Genomics Vol. 16 Journal Issue: 1; ISSN 1471-2164
Publisher:
Springer Science + Business MediaCopyright Statement
Country of Publication:
United Kingdom
Language:
English
Citation Metrics:
Cited by: 62 works
Citation information provided by
Web of Science

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Conducting metagenomic studies in microbiology and clinical research journal August 2018
Next-generation sequencing and waste/wastewater treatment: a comprehensive overview journal September 2019
Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis journal August 2018
Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries journal July 2018
Next-generation physiology approaches to study microbiome function at single cell level journal February 2020
Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies journal November 2019
Sources and selection of snow-specific microbial communities in a Greenlandic sea ice snow cover journal February 2019
Next generation sequencing data of a defined microbial mock community journal September 2016
Issues and current standards of controls in microbiome research journal April 2019
GC bias affects genomic and metagenomic reconstructions, underrepresenting GC-poor organisms journal February 2020
Bacteria in the ageing gut: did the taming of fire promote a long human lifespan? journal June 2018
Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF journal September 2018
Studying the gut virome in the metagenomic era: challenges and perspectives journal October 2019
A pipeline for targeted metagenomics of environmental bacteria journal February 2020
Usability of rectal swabs for microbiome sampling in a cohort study of hematological and oncological patients journal April 2019
Mycobiome Sequencing and Analysis Applied to Fungal Community Profiling of the Lower Respiratory Tract During Fungal Pathogenesis journal March 2019
Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters journal May 2019
BinSanity: unsupervised clustering of environmental microbial assemblies using coverage and affinity propagation journal January 2017
Optimizing de novo genome assembly from PCR-amplified metagenomes journal January 2019

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59 BASIC BIOLOGICAL SCIENCES
Low input
Low biomass
Metagenome
Library preparation protocol
Microbiome