SMRT-Tag prepartion of Homo sapiens: HG002 NIST reference material
Genomic DNA is tagmented using a custom-loaded Tn5 transposase with hairpin adapters compatible with PacBio SMRT sequencing in 10mM TAPS-NaOH pH 7.5, 5mM MgCl2, and 10% DMF at 55C for 30 minutes. Fragments are cleaned up using a 2X SPRI and gap-repaired using a polymerase / ligase combination (Phusion polymerase / Taq ligase or T4 DNA polymerase / Ampligase) at 37C for 1hr. Libraries are then digested with exonuclease III at 37C for 1hr to remove un-repaired, non-productive templates. Libraries were optionally size selected for fragments > 5kb with a 3.1X cleanup using diluted (35% v/v) Ampure PB, and the low molecular weight fraction optionally recovered for additional sequencing.