Pulmonary tissue sample 11
DNA was extracted from lung tissue samples by a modified version of the Qiagen DNeasy kit.  The extracted DNA was normalized by QPCR (515F/1391R) and then preparatively amplified in 50 microliter reactions using barcoded Illumina 'fusion' primers targeting the 515F/907R amplicon and the thermal program: 94 C, 5 min; then 20 – 24 cycles of (92 C, 20 s; 50 C, 30 s; 65 C, 60 s; 75 C, 60 s; 70 C, 60 s) with an additional 10 min, 70 C final extension step.  These libraries were subsequently converted for compatibility with the Ion Torrent PGM using appropriate 515F/907R fusion primers in 6 - 10 cycles of PCR under the same conditions.  Purified products were quantified by real-time PCR using primers complementary to the adapter sequences.