Sequencing for C6 (Peru, 1991)
To generate SMRTbell libraries, genomic DNA was mechanically sheared to a target size of 2,000 base pairs using a Covaris™ S2 System (Covaris, Woburn, MA) according to the manufacturer’s instructions.  Sheared samples were then concentrated and size-selected (i.e., fragments greater than 100 bp were retained) by Solid Phase Reversible Immobilization (SPRI) paramagnetic bead based technology (AMPure XP ™, Beckman-Coulter, Danvers, MA).  Samples were eluted in a final volume of 20 uL 10mM Tris pH 7.5, with concentrations ranging from 0.5 to 1 ug/uL (Nanodrop, Thermo Scientific, Wilmington, DE).  Fragment size distributions were verified by standard agarose gel electrophoresis (Flashgel, Lonza Rockland, Inc.  Rockland, ME), and using an Agilent 2100 Bioanalyzer with DNA 7500 chemistry (Agilent, Santa Clara, CA).  The means from the fragment distributions were between 1700-2000 bp.  Blunt ends were generated in an end-polishing reaction, and the 5’ends simultaneously phorsphorylated.  Universal hairpin adapters were ligated onto the ends of the prepared DNA fragments.   Hairpin adapters are single-stranded oligonucleotide loops of sequence 5’–pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT–3’.  Prior to ligation, hairpins were diluted to 20 uM in the presence of 10 mM Tris (pH 7.5) and 100mM NaCl and annealed into a stem-loop structure.  Annealed hairpins were added at ~30X molar excess relative to the insert and ligated using T4 DNA ligase (NEB).  Failed ligation products are removed through the use of exonucleases as previously described33.  Hairpin dimers formed during ligation were removed in a final magnetic bead purification (double-SPRI) with size-selective conditions (i.e., SMRTbells >500bp were retained).  Purified SMRTbell libraries were eluted in a final volume of 15uL 10mM Tris pH 7.5, with concentrations between 0.1 – 0.25 ug/uL (Nanodrop, Thermo Scientific, Wilmington DE).   SMRTbell size distributions were determined using an Agilent 2100 Bioanalyzer with DNA 7500 chemistries (Agilent, Santa Clara, CA).  SMRTbell libraries were centered on a mean of 1800-2100 bp.   SMRTbells were annealed to sequencing primer complementary to a sequence in the hairpin loop (5’– AACGGAGGAGGAGGA –3’) at a final concentration of 40nM DNA template.