PET (Paired End-Tag) Genomic Shotgun Library Construction Protocol

This protocol begins by taking the genomic DNA to be sequenced, and shearing it randomly with a tight size distribution (typical length ~ 1.0 kbp), using a Hydroshear or equivalent device. The sheared genomic DNA is then size selected, end repaired, A-tailed, and circularized with a T-tailed 30 bp long synthetic oligonucleotide (T30). The resulting circular DNA undergoes rolling circle amplification, and is then digested with Mme1, resulting in 70 bp components consisting of the synthetic T30 sequence, flanked by two 17-18 bp tags of genomic DNA. The 70 bp paired insert libraries are then size selected and end repaired, and two flanking ePCR primer oligonucleotides (FDV and RDV) are ligated to their 3’ and 5’ ends. The resulting 135 bp library molecule is size selected, nick translated, PCR amplified, and (optionally) size selected. The final result is a paired-insert library of 133-135 bp template DNA, consisting of a 44 bp FDV sequence, a 17-18 bp proximal tag, the T30 sequence, a 17-18 bp distal tag, and a 25 bp RDV sequence.