Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

Abstract

Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1–V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacer and Enterococcus predominated in the community generated by V4–V6 primers, and the most numerous genera in the V7–V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4–V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7–V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1–V3 and V7–V9 primers providesd results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.

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.…”

Patient-specific Analysis of Periodontal and Peri-implant Microbiomes

“…Two regions of the 16S rRNA genes were sequenced: V1-V3 and V7-V9. The primers used for sequencing have been previously described (Kumar et al, 2011). Adaptor sequences were trimmed from raw data with 98% or more of bases demonstrating a quality control of 30, and sequences were binned into individual sample collections based on bar-code sequence tags, which were then trimmed.…”

River bacterioplankton community responses to a high inflow event

“…DNA samples were amplified with the 16S rRNA universal Eubacterial primers 27F (5-AGA GTT TGA TCC TGG CTC AG-3') and 519R (5-GTN TTA CNG CGG CKG CTG-3') (Kim et al 2011, Kumar et al 2011) and a PCR reaction using the HotStarTaq Plus Master Mix Kit (Qiagen) using the following cycling conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s; 53°C for 40 s and 72°C for 1 min; after which a final elongation step at 72°C for 5 min was performed. Post-PCR purification was performed using Agencourt Ampure beads (Agencourt Bioscience).…”