title: Comprehensive Long Span Paired-End-Tag Mapping Reveals Recurrent Fusion Genes in Gastric Cancer Genomes
design: DNA-PET libraries were constructed for each sample, eight patients' primary tumor and paired blood samples as follows: genomic DNA was hydrosheared to 7-12 kb DNA fragments. Long Mate Paired (LMP) cap adaptors were ligated to the hydrosheared and end repaired DNA fragments. The cap adaptor-ligated DNA fragments were separated by agarose gel electrophoresis, recovered using the QIAEXII Gel Extraction Kit (QIAGEN) and circularized with a biotinylated adaptor that connects the cap adaptors at both ends of the DNA fragments. Missing 5’ phosphate groups of cap adaptors created a nick on each strand after circularization of the DNA. Both nicks were translated outwards by >50 bp into the circularized genomic DNA fragment by DNA polymerase I (NEB). The nick-translated constructs were then digested with T7 exonuclease and S1 nuclease (NEB), to release paired-end tag (PETs) library constructs. These constructs were ligated with SOLiD sequencing adaptors P1 and P2 (Life Technologies), and amplified using 2x HF Phusion Master Mix (Finnzymes OY) for sequencing.  High throughput sequencing of the 2x50 bp libraries was performed on SOLiD sequencers (v3plus and v4, respectively) according to the manufacturer’s recommendation (Life Technologies). Sample CD15 has been sequenced by 2x35 bp. Sequence tags were mapped to the human reference sequence (NCBI Build 36) and paired using SOLiD System Analysis Pipeline Tool Bioscope, allowing up to 12 color code mismatches per 50 bp tag and up to 4 color code mismatches per 35 bp tag. For sample CD5 and CD8, two DNA size fractions were merged for library construction which resulted in a reduced sensitivity to identify small deletions.