P-GSE42426-1 - P-GSE42426-1
nucleic acid library construction protocol
Skin biopsies of 6 mm were taken from the centre of the lesion using a sterile biopsy punch. The biopsies were cut in small pieces, suspended in 0.5 ml of sterile PBS and frozen at -20 ºC. Bacterial DNA was extracted from tissue samples (approximately 2mm2) using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). First, the biopsy samples were incubated for 1.5 h at 55°C in 180 µl lysis buffer with 20 µl proteinase K provided in the kit. All subsequent steps were performed according to the instructions provided by the manufacturer. For each sample, 2 µl of template DNA was applied for the construction of sequencing libraries. DNA was amplified by PCR using the Treponema specific primers designed for this study. Each sample was amplified with a unique primer with an added hexamer barcode at the 5’ end of the forward and reverse primer. Amplification of the target region was carried out in a 50 µl reaction mix which contained 1 × AmpliTaq buffer (Applied Biosystems, Carlsbad, CA), 100 µM of each deoxynucleoside triphophate (Amersham Biosciences, Piscataway, NJ), 0.2 pmol of each primer, 2.5 U of AmpliTaq DNA polymerase (Applied biosystems), and 2 µl of template DNA. Thermal cycling using a T3 Thermocycler (Biometram Göttingen, Germany) was performed as follows: denaturation at 94°C for 3 min and 30 s, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 59°C for 45 s, and extension at 72°C for 1 min, followed by a final elongation step of 5 min. Equal amounts of all 40 amplicons were pooled (final concentration of 5 µg) and purified with the Qiagen Mini Elute kit (Qiagen), according to the protocol provided by the manufacturer. Next generation sequencing was performed by LCG Genomics (Teddington, Middlesex, UK) using Roche / 454 Genome Sequencer FLX + Titanium according to their standard procedure (1/4 PLP).