Cystic Fibrosis Subject 5
DNA was extracted from sputum 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 'fusion' primers targeting the 515F/1391R 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.  Purified products were quantified by real-time and digital QPCR using primers complementary to the 454 adapter sequences.