High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.
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.
Centre for Systems Biology, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
Martin J
MJ
Sergeant
Chrystala
C
Constantinidou
Tristan
T
Cogan
Charles W
CW
Penn
Mark J
MJ
Pallen
D000818:Animals; D002432:Cecum; D002645:Chickens; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013696:Temperature
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D004247:DNA