Short pyrosequencing reads suffice for accurate microbial community analysis

Abstract

Pyrosequencing technology allows us to characterize microbial communities using 16S ribosomal RNA (rRNA) sequences orders of magnitude faster and more cheaply than has previously been possible. However, results from different studies using pyrosequencing and traditional sequencing are often difficult to compare, because amplicons covering different regions of the rRNA might yield different conclusions. We used sequences from over 200 globally dispersed environments to test whether studies that used similar primers clustered together mistakenly, without regard to environment. We then tested whether primer choice affects sequence-based community analyses using UniFrac, our recently-developed method for comparing microbial communities. We performed three tests of primer effects. We tested whether different simulated amplicons generated the same UniFrac clustering results as near-full-length sequences for three recent large-scale studies of microbial communities in the mouse and human gut, and the Guerrero Negro microbial mat. We then repeated this analysis for short sequences (100-, 150-, 200- and 250-base reads) resembling those produced by pyrosequencing. The results show that sequencing effort is best focused on gathering more short sequences rather than fewer longer ones, provided that the primers are chosen wisely, and that community comparison methods such as UniFrac are surprisingly robust to variation in the region sequenced.

Antimicrobial and antioxidant activities of Enterococcus species isolated from meat and dairy products

“…The V3 region of 16S rRNA gene is a commonly used region for bacterial phylogenetic analysis. Despite of the short nucleic acid sequences, hypervariable V3 region can provide information enough to describe microbial community (Liu et al, 2007). The isolates were clustered within E. faecium species, and the results obtained by 16S rRNA sequencing indicated a high degree of sequence similarity among them and from a phylogenetically coherent group of lactic acid bacteria.…”

Microbial biodiversity of the liquid fraction of rumen content from lactating cows

“…The primers used for the Rumen microbiome biodiversity in lactating cows Eubacteria 16S rRNA amplification were F8 AGAGTTTG ATCCGGCTCAC (Baker et al, 2003) and R534 ATTACCG CGGCTGCTGGC (Liu et al, 2007), which produced amplicons of about 500 base pairs (bps). The primers were chosen to amplify the eubacteria 16S rRNA regions V1-V3, as reported by Liu et al (2007 and, as being the most reliable to produce community clusters and to accurately assign taxonomy in a large data set (Kumar et al, 2011;Vilo and Dong, 2012). The primers above were added with multiplex identifier tags and 454 adapters A and B to allow pooling of the amplicons generated from different individuals before sequencing.…”

Effect of pH on soil bacterial diversity

“…A 20 ng aliquot of the 16S rDNA gene from the extracted DNA samples was amplified using universal primer sets, 27F (5′-GAGTTTGAT CMTGGCTGG-3′) primer with a Roche 454 A pyrosequencing adapter and 518 R (5′-GTATTACCGCG GCTGCTGG-3′) with a Roche 454 B sequencing adapter. The targeted gene region is believed as the most reliable for the accurate taxonomic classification of bacterial sequences (Liu et al 2007). Pyrosequencing library was prepared using PCR products according to the GS FLX titanium library prep guide.…”