Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample

Abstract

The ongoing revolution in high-throughput sequencing continues to democratize the ability of small groups of investigators to map the microbial component of the biosphere. In particular, the coevolution of new sequencing platforms and new software tools allows data acquisition and analysis on an unprecedented scale. Here we report the next stage in this coevolutionary arms race, using the Illumina GAIIx platform to sequence a diverse array of 25 environmental samples and three known "mock communities" at a depth averaging 3.1 million reads per sample. We demonstrate excellent consistency in taxonomic recovery and recapture diversity patterns that were previously reported on the basis of metaanalysis of many studies from the literature (notably, the saline/ nonsaline split in environmental samples and the split between host-associated and free-living communities). We also demonstrate that 2,000 Illumina single-end reads are sufficient to recapture the same relationships among samples that we observe with the full dataset. The results thus open up the possibility of conducting large-scale studies analyzing thousands of samples simultaneously to survey microbial communities at an unprecedented spatial and temporal resolution.human microbiome | microbial community analysis | microbial ecology | next-generation sequencing H igh-throughput sequencing technologies have opened new frontiers in microbial community analysis by providing a cost-effective means of identifying the microbial phylotypes that are present in samples. These studies have revolutionized our understanding of the microbial communities in our bodies (1, 2) and on our planet (3-5). This revolution in sequencing technology, combined with the development of advanced computational tools that exploit metadata to relate hundreds of samples to one another in ways that reveal clear biological patterns, has reinvigorated studies of the 16S rRNA gene (6

Heart failure is associated with depletion of core intestinal microbiota

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Eggshells as hosts of bacterial communities: An experimental test of the antimicrobial egg coloration hypothesis

“…We confirmed the success of DNA extractions by PCR with primers 515F and 806R which amplify the V4 variable region of the 16S rRNA of bacteria (Caporaso et al., 2011). Reaction components were 1.5 μl 10× GeneAmp buffer, 1.2 μl 25 mmol/L MgCl 2 , 1.5 μl 2 mmol/L dNTP, 0.4 μl 10 μmol/L primer 515F, 0.4 μl 10 μmol/L primer 806R, 6.175 μl water, 0.075 μl AmpliTaq Gold DNA polymerase, and 3.75 μl template DNA.…”

Plant community and soil conditions individually affect soil microbial community assembly in experimental mesocosms

“…Total DNA was extracted from ~0.25 g soil using the PowerSoil DNA extraction kit (Mo Bio Laboratories, Inc.) following manufacturer's instructions. The V4 region of the 16s rRNA gene was amplified in triplicate using custom barcoded primers (515f/806r primer pair; Caporaso et al., 2011) then pooled in equimolar ratios, cleaned, and quantified following published protocols (Caporaso et al., 2011, 2012; Maurice, Haiser, & Turnbaugh, 2013). All samples were sequenced in a single MiSeq lane with paired‐end 250 bp reads and reagent kit v2 (Illumina, San Diego, CA).…”