Abstract
BackgroundDNA-stable isotope probing (DNA-SIP) links microorganisms to their in-situ function in diverse environmental samples. Combining DNA-SIP and metagenomics (metagenomic-SIP) allows us to link genomes from complex communities to their specific functions and improves the assembly and binning of these targeted genomes. However, empirical development of metagenomic-SIP methods is hindered by the complexity and cost of these studies. We developed a toolkit, ‘MetaSIPSim,’ to simulate sequencing read libraries for metagenomic-SIP experiments. MetaSIPSim is intended to generate datasets for method development and testing. To this end, we used MetaSIPSim generated data to demonstrate the advantages of metagenomic-SIP over a conventional shotgun metagenomic sequencing experiment.ResultsThrough simulation we show that metagenomic-SIP improves the assembly and binning of isotopically labeled genomes relative to a conventional metagenomic approach. Improvements were dependent on experimental parameters and on sequencing depth. Community level G + C content impacted the assembly of labeled genomes and subsequent binning, where high community G + C generally reduced the benefits of metagenomic-SIP. Furthermore, when a high proportion of the community is isotopically labeled, the benefits of metagenomic-SIP decline. Finally, the choice of gradient fractions to sequence greatly influences method performance.ConclusionsMetagenomic-SIP is a valuable method for recovering isotopically labeled genomes from complex communities. We show that metagenomic-SIP performance depends on optimization of experimental parameters. MetaSIPSim allows for simulation of metagenomic-SIP datasets which facilitates the optimization and development of metagenomic-SIP experiments and analytical approaches for dealing with these data.
Highlights
DNA-stable isotope probing (DNA-Stable isotope probing (SIP)) links microorganisms to their in-situ function in diverse environmental samples
We demonstrated the utility of MetaSIPSim by assessing whether the coverage, assembly, and Metagenome assembled genome (MAG) binning of target 13C-labeled genomes were improved by metagenomicSIP relative to a conventional shotgun metagenomic approach
We found that the peak genomic DNA density recovery from the MetaSIPSim and SIPSim simulations roughly matched the empirical results from Buckley et al 2007 [25] and Wawrik et al 2009 [35] (Fig. 2b and c) and the general distributions were similar across datasets for these studies
Summary
DNA-stable isotope probing (DNA-SIP) links microorganisms to their in-situ function in diverse environmental samples. Combining DNA-SIP and metagenomics (metagenomic-SIP) allows us to link genomes from complex communities to their specific functions and improves the assembly and binning of these targeted genomes. MetaSIPSim is intended to generate datasets for method development and testing To this end, we used MetaSIPSim generated data to demonstrate the advantages of metagenomic-SIP over a conventional shotgun metagenomic sequencing experiment. DNA-Stable isotope probing (DNA-SIP) is a powerful tool for linking uncultured microorganisms to their function within environmental samples [1]. Validation and improvement of metagenomic-SIP methods and analytical tools have largely been hindered by the difficulty and cost of these experiments [14, 20]
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