Illumina Genome Analyzer IIx paired end sequencing; Paired end sequencing of PEO4
Low-coverage paired-end sequencing of PEO4
Genomic DNA was extracted from a confluent T75 using the Gentre Puregene cell kit for 1-2 e7 cells (Qiagen, Crawley, UK) according to manufacturer’s instructions. 5ug was used for library construction. All equipment was washed with MilliQ water, cleaned with DNA Away wipes (Molecular Bioproducts) and UV treated prior to making each library to avoid cross-contamination. Target capture was carried out using the SureSelect Human AllExon kit (Agilent) according to manufacturer’s instructions but with minor modifications. Sonication was performed in a 250ul volume using a Bioruptor, pulsing for 30 seconds on, 30 seconds off, at high power, with a total sonication time of 45 minutes.Samples were processed using the Illumina paired-end sample prep kit (Illumina, UK) according to the manufacturer’s protocol with minor alterations. Samples were fragmented in a volume of 200ul 1xTE, using a Bioruptor pulsing for 30 seconds on, 30 seconds off, at high power, with a total sonication time of 10 minutes. Samples were cleaned up using a Qiagen PCR purification kit and eluted in 30ul EB. 10 ul loading dye was added and samples were run on a 2% TAE agarose gel stained with Sybr safe (Invitrogen) to obtain a smear of 100-1000 bp. A section of gel approximately 2mm wide centred at 400 bp was excised. DNA was recovered from the gels using a Qiagen gel extraction kit and eluting in 3ul EB. End repair and the addition of A overhangs was performed according to manufacturer’s instructions. For adapter ligation PE adapter oligo mix input volume was reduced to 2ul, to reflect the loss of DNA during the first gel extraction. The volume for the ligation reaction was made up to 50ul by the addition of 8ul nuclease free water. Ligation products were gel separated according to the Illumina protocol and a thin gel slice corresponding to approximately 400 bp was excised. Following gel extraction the library was PCR amplified. The quantity of input DNA was increased to 10ul in order to reduce the number of PCR cycles required and therefore the number of duplicates sequenced. 10 cycles of PCR were performed before the amplified libraries were purified using Qiagen PCR purification kit and eluted in 30ul EB. Library quality and insert size was checked on the Bioanalyzer before loading onto a flow cell.