A standardized quantitative analysis strategy for stable isotope probing metagenomics
[4];
[1];
[1];
[2];
[1];
- DOE Joint Genome Institute, Lawrence Berkeley National Laboratory , Berkeley, California, USA
- Department of Civil Engineering, The University of British Columbia , Vancouver, British Columbia, Canada
- Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory , Livermore, California, USA
- Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory , Livermore, California, USA, Life &, Environmental Sciences Department, University of California Merced , Merced, California, USA
Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential.
- Research Organization:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE National Nuclear Security Administration (NNSA)
- Grant/Contract Number:
- AC02-05CH11231; RGPIN-2018-04585; SWC1632; AC52-07NA27344
- OSTI ID:
- 1987570
- Alternate ID(s):
- OSTI ID: 1990056; OSTI ID: 2282835
- Report Number(s):
- LLNL-JRNL-852817; e01280-22
- Journal Information:
- mSystems, Journal Name: mSystems; ISSN 2379-5077
- Publisher:
- American Society for MicrobiologyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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