Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics - PubMed (original) (raw)

Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics

Bonnie L Hurwitz et al. Environ Microbiol. 2013 May.

Free PMC article

Abstract

Viruses have global impact through mortality, nutrient cycling and horizontal gene transfer, yet their study is limited by complex methodologies with little validation. Here, we use triplicate metagenomes to compare common aquatic viral concentration and purification methods across four combinations as follows: (i) tangential flow filtration (TFF) and DNase + CsCl, (ii) FeCl3 precipitation and DNase, (iii) FeCl3 precipitation and DNase + CsCl and (iv) FeCl3 precipitation and DNase + sucrose. Taxonomic data (30% of reads) suggested that purification methods were statistically indistinguishable at any taxonomic level while concentration methods were significantly different at family and genus levels. Specifically, TFF-concentrated viral metagenomes had significantly fewer abundant viral types (Podoviridae and Phycodnaviridae) and more variability among Myoviridae than FeCl3 -precipitated viral metagenomes. More comprehensive analyses using protein clusters (66% of reads) and k-mers (100% of reads) showed 50-53% of these data were common to all four methods, and revealed trace bacterial DNA contamination in TFF-concentrated metagenomes and one of three replicates concentrated using FeCl3 and purified by DNase alone. Shared k-mer analyses also revealed that polymerases used in amplification impact the resulting metagenomes, with TaKaRa enriching for 'rare' reads relative to PfuTurbo. Together these results provide empirical data for making experimental design decisions in culture-independent viral ecology studies.

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

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Figures

Fig. 1

Fig. 1

Workflow showing the process of creating each viral metagenomic replicate using different concentration (TFF or FeCl3) and purification methods (DNase only, DNase + CsCl or DNase + sucrose) from a 210 l sample of seawater taken from Scripps Pier, San Diego, CA. The ‘3 ×’ refers to that each subsample was processed independently from the 210 l pooled initial sample.

Fig. 2

Fig. 2

Rank abundance curve of (A) family-level (B) and genus-level taxonomy for the top 10 taxa observed in the data. Only four family-level and four genus-level taxa are viral (highlighted with red text); the remaining are microbial taxa.

Fig. 3

Fig. 3

Rarefaction analysis of hits to protein clusters from each viral metagenome using (A) all sequences and (B) abundant (k-mer > 1) sequences. To be conservative, only protein clusters with > 20 members were used in these analyses.

Fig. 4

Fig. 4

K-mer-based analysis of shared reads between methods and replicates. (A) Percentage of total reads that are shared between methods based on a k-mer-based analysis and (B) percentage of ‘rare’ (k-mer = 1) versus abundant sequences (k-mer > 1) in each of the four viral metagenomic methods and a non-replicated microbial metagenome. The enzymes used for linker amplification (TaKaRa or PfuTurbo) are listed above each sample. The microbial sample includes more ‘rare’ sequences because the diversity in the sample is undersampled based upon rarefaction analysis (data not shown).

Fig. 5

Fig. 5

Superkingdom taxonomic profile of reads for each triplicate sample from the four viral metagenome methods and a non-replicated microbial metagenome for (A) rare (k-mer = 1) and (B) abundant (k-mer > 1) reads.

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