title: Comparison of methods to identify pathogens and associated virulence functional genes in biosolids from two different wastewater treatment facilities in Canada
design: For the 16S rRNA gene, eight libraries were prepared using two different universal bacterial primer sets on four different samples (“Plant A August 2009 before-after” and “Plant C May 2009 before-after”). The following primers were used: V1-V3: forward  5’ CGTATCGCCTCCCTCGCGCCATCAGACGAGTGCGTAGTTTGATCCTGGCTCAG-3’, reverse 5’- CTATGCGCCTTGCCAGCCCGCTCAGACGCTCGACACATTACCGCGGCTGCTGG-3’ and V3-5: forward 5’- CGTATCGCCTCCCTCGCGCCATCAGAGACGCACTCGCCTACGGGAGGCAGCAG-3’ reverse 5’- CTATGCGCCTTGCCAGCCCGCTCAGAGCACTGTAGCCGTCAATTCMTTTRAGT-3’ where the italic sequence represents the sample specific multiplex identifier, the bold sequence represents the template specific sequences and the remaining sequence is the 454 adapter A (forward) and adapter B (reverse).   For the cpn60   gene, four libraries were prepared using the “Plant A August 2009 before-after” and “Plant C May 2009 before-after” samples. The following universal primers were used: for “Plant A August 2009  before” and “Plant C May 2009 before”, forward: 5’- CGTATCGCCTCCCTCGCGCCATCAGATCAGACACGGCIGGIGAYGGNACNACNAC3’, reverse : 5’- CTATGCGCCTTGCCAGCCCGCTCAGATATCGCGAGTCICCRAANCCNGGNGCYTT-‘3. The same primers were used for “Plant A August 2009 after” and “Plant C May 2009 after” except that the multiplex identifiers (in italics) were replaced by the following: forward primer: CGTGTCTCTA and reverse primer: CTCGCGTGTC.  The PCR conditions were as follows: the mixture in a 50ul final volume contained 50ng DNA, 25pmol of each primer for 16S or cpn60, 1X final Taq polymerase buffer, and 2.5 units of Taq polymerase (New England BioLabs Ltd, Pickering, ON, Canada) and 1ul of 10 mM deoxynucleoside triphosphates. PCR cycling consisted of 94oC for 5 minutes, denaturation at 94oC for 30 seconds, annealing at 56oC for 30 seconds for 16S V3-1, 50oC for 16S V5-3 and 55oC for cpn60  , with an extension at 72oC for 45 seconds after 35 cycles. A final extension at 72oC was added for 7 minutes.