Noise and the accurate determination of microbial diversity from 454 pyrosequencing data
A 5 litre sample of Priest Pot lake water was collected in winter, transported on ice and filtered  through a 0.2µm membrane filter to concentrate the microorganisms. Microbial biomass  was washed from the 0.2µm filter using the 50mL of the filtrate and the DNA was extracted  using the FastDNA kit for soils according to the manufacturer's instructions (MP Biomedicals,  Cambridge, UK).    This sample was then amplified using modified primers 787f and 1492r, with attached 454 adaptors A and B respectively at the 5' end.The oligonucleotide sequences used were A-787f, 5'-gcctccctcgcgccatcagATTAGATACCCNGGTAG-  3', and B-1492r, 5'-gccttgccagcccgctcagGNTACCTTGTTACGACTT-3', where 454 Life Science's  A or B adaptors are shown in lowercase. The PCR conditions were 95oC for 5 minutes,  30 cycles of: 95oC for 30 seconds; 57oC for 30 seconds; 72oC for 1 minute, and finally 72oC  for 7 minutes. The polymerase used was standard Taq. Several PCR amplifications were  carried out in order to obtain a total of 500ng of DNA for pyrosequencing, the bulked products  of which were cleaned-up using a QIAquick PCR purification kit according to the manufacturer's  instructions. Pyrosequencing of these amplicons was then performed from the A adaptor and a total of 28361 reads obtained.