Genomic Epidemiology of a Tuberculosis Outbreak: MT0030
Illumina Paired End DNA Sample Prep protocol (PE-102-1001) is used to build DNA libraries with insert sizes from 160-200 base pairs for paired-end sequencing. 3ug DNA was sheared by sonication and run on 8% PAGE gel. The 200bp DNA fraction was excised and eluted from the gel slice and was purified using a Spin-X Filter Tube (Fisher Scientific) and by ethanol precipitation. Library was prepared using a modified paired-end protocol supplied by Illumina Inc. This involved DNA end-repair and addition of 3' adenosine overhangs using the Klenow fragment of DNA polymerase I (3'–5' exonuclease minus) and ligation to Illumina PE adaptors. Adaptor-ligated products were purified and PCR-amplified using Phusion DNA polymerase (NEB) for 10 or 15 cycles with the PE primer 1.0 and 2.0 (Illumina). PCR products of the desired size range were purified from adaptor ligation artifacts using 8% PAGE gel. Library DNA quality was assessed using an Agilent DNA 1000 series II assay (Agilent) and DNA was subsequently diluted to 10 nM. The final concentration was confirmed using a Quant-iT dsDNA HS assay kit and Qubit fluorometer (Invitrogen).