Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community.
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 composition 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.
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. rmbowers@lbl.gov.
Robert M
RM
Bowers
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. aclum@lbl.gov.
Alicia
A
Clum
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. hundley5@llnl.gov.
Hope
H
Tice
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. jhlim@lbl.gov.
Joanne
J
Lim
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. ksingh@lbl.gov.
Kanwar
K
Singh
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. dgciobanu@lbl.gov.
Doina
D
Ciobanu
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. cyngan@lbl.gov.
Chew Yee
CY
Ngan
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. jfcheng@lbl.gov.
Jan-Fang
JF
Cheng
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. sgtringe@lbl.gov.
Susannah G
SG
Tringe
Microbial Genomics Program Lead, DOE Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA, USA. twoyke@lbl.gov.
Tanja
T
Woyke
D001105:Archaea / Q000235:genetics; D001419:Bacteria / Q000235:genetics; D001482:Base Composition; D018533:Biomass; D020451:Contig Mapping; D015723:Gene Library; D054892:Metagenome; D056186:Metagenomics / Q000379:methods; D064307:Microbiota; D017422:Sequence Analysis, DNA