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High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

Sergeant MJ, Constantinidou C, Cogan T, Penn CW, Pallen MJ - PLoS ONE (2012)

Bottom Line: Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased.Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing.In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

View Article: PubMed Central - PubMed

Affiliation: Centre for Systems Biology, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

ABSTRACT
The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

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Rarefaction curves of the 97% OTUs for the different experimental protocols.At each sampling depth, the average number of OTUs is shown (n=2) (a) Different primer pairs (b) Different annealing temperatures (c) Different extraction procedures.
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pone-0038094-g001: Rarefaction curves of the 97% OTUs for the different experimental protocols.At each sampling depth, the average number of OTUs is shown (n=2) (a) Different primer pairs (b) Different annealing temperatures (c) Different extraction procedures.

Mentions: Use of short primers had little effect on species richness (Figure 1a) or species evenness as measured by Simpsons' Index (Table 1), nor did their use lead to the identification of any significant novel OTUs. However, there were substantial differences in relative abundance of OTUs (Table 3) and the community structures of the libraries created by the different primer combinations were clearly different as judged by UGPMA clustering (Figure 2a) and PCA analysis (Figure 3a). In particular, Bifidobacterium could not be detected at all by the shorter forward primers, but could be detected at higher abundance using the shorter reverse primer. In addition OTUs corresponding to Lactobacillus were more abundant when the shorter reverse primer was used.


High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

Sergeant MJ, Constantinidou C, Cogan T, Penn CW, Pallen MJ - PLoS ONE (2012)

Rarefaction curves of the 97% OTUs for the different experimental protocols.At each sampling depth, the average number of OTUs is shown (n=2) (a) Different primer pairs (b) Different annealing temperatures (c) Different extraction procedures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362549&req=5

pone-0038094-g001: Rarefaction curves of the 97% OTUs for the different experimental protocols.At each sampling depth, the average number of OTUs is shown (n=2) (a) Different primer pairs (b) Different annealing temperatures (c) Different extraction procedures.
Mentions: Use of short primers had little effect on species richness (Figure 1a) or species evenness as measured by Simpsons' Index (Table 1), nor did their use lead to the identification of any significant novel OTUs. However, there were substantial differences in relative abundance of OTUs (Table 3) and the community structures of the libraries created by the different primer combinations were clearly different as judged by UGPMA clustering (Figure 2a) and PCA analysis (Figure 3a). In particular, Bifidobacterium could not be detected at all by the shorter forward primers, but could be detected at higher abundance using the shorter reverse primer. In addition OTUs corresponding to Lactobacillus were more abundant when the shorter reverse primer was used.

Bottom Line: Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased.Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing.In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

View Article: PubMed Central - PubMed

Affiliation: Centre for Systems Biology, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

ABSTRACT
The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

Show MeSH