Analysis of 16S rRNA Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform

Abstract

Background16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platformMethodology/Principal FindingsThe following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.ConclusionsOur findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

The effects of initial substrate concentration, C/N ratio, and temperature on solid-state anaerobic digestion from composting rice straw

“…Total DNA was extracted according to the method described previously (Rademacher et al, 2012). For sequencing, the 16S rRNA genes were amplified with primers 515F (5 0 -GTGYCAGCMGCCGCGGTA-3 0 ) and 909R (5 0 -CCCC GYCAATTCMTTTRAGT-3 0 targeting the bacterial and archaeal V4-V5 region in the 16S rRNA gene) with a 10-mer barcode at the 5 0 end of primer 515F (Tamaki et al, 2011). To minimize PCR bias, two PCR reactions were set up for each sample, and the PCR products in the replicate reactions were pooled.…”

Straw- and slurry-associated prokaryotic communities differ during co-fermentation of straw and swine manure

“…For pyrosequencing, the 16S rRNA genes were amplified with primers 515F (5′-GTGYCAGCMGCCGCGGTA-3′) and 909R (5′-CCCC GYCAATTCMTTTRAGT-3′) (targeting bacterial and archaeal V4-V5 region in 16S rRNA gene) with a 10-mer barcode at the 5′ end of primer 515F (Tamaki et al 2011). In order to minimize PCR bias, two PCR reactions were set up for each sample, and the PCR products in the replicate reactions were pooled.…”

Rumen prokaryotic communities of ruminants under different feeding paradigms on the Qinghai-Tibetan Plateau

“…Extracted DNA was diluted to 20 ng L −1 and stored at −40 • C for downstream use. For MiSeq sequencing, the V4-V5 hypervariable region of 16S rRNA genes was amplified with universal primers 515F (5 -GTGCCAGCMGCCGCGGTAA-3 ) and 909R (5 -CCCCGYCAATTCMTTTRAGT-3 ) [74]. The PCR amplification program included initial denaturation at 94 • C for 3 min, followed by 30 cycles of 94 • C for 40 s, 56 • C for 60 s, and 72 • C for 60 s, and a final extension at 72 • C for 10 min.…”