Ashkenazim trio- Son_NA24385-PacBio -P6 chemistry
"DNA library preparation and sequencing was performed according to the manufacturer's  instructions with noted modifications. Following the Pacific Biosciences Protocol,  ""20-kb Template Preparation Using Blue Pippin Size-Selection System"", library  preparation was performed using the Pacific Biosciences SMRTbell Template Prep Kit 1.0 (PN # 100-259-100).  In short, 10 µg of extracted, high-quality, genomic DNA from eac of HG-002, HG-003, and HG-004, the AJ trio, were used for library preparation. Genomic DNA extracts were verified with the Life Technologies Qubit 2.0 Fluorometer using the High Sensitivity dsDNA assay (PN# Q32851) to quantify the mass of double-stranded DNA present.  After quantification, each sample was diluted to 150 µL, using kit provide EB, yielding a concentration of ~ 66 ng/µL.  The 150 µL aliquots were individuall pipetted into the top chambers of Covaris G-tube (PN# 520079) spin columns and sheared  for 60 seconds at 4500 rpm using an Eppendorf 5424 benchtop centrifuge. Once complete,  the spin columns were flipped after verifying that all DNA was now in the lower chamber.  The columns were spun for another 60 seconds at 4500 rpm to further shear the DNA and  place the aliquot back into the upper chamber. In some cases G-tubes were centrifuged  2-3 times, in both directions to ensure all volume had passed into the appropriate  chamber.  Shearing resulted in a ~20,000 bp DNA fragments verified using an Agilent  Bioanlyzer DNA 12000 gel chip (PN# 5067-1508).  The sheared DNA isolates were then  purified using a 0.5X AMPure PB magnetic bead purification step (0.5X AMPure PB beads  added, by volume, to each DNA sample, vortexed for 10 minutes at 2,000 rpm, followed  by two washes with 70% alcohol and finally eluted in EB). This AMPure purification  step assures removal of any small fragment and/or biological contaminant.  The sheared  DNA concentration was then measured using the Qubit High Sensitivity dsDNA assay.   These values were used to calculate actual input mass for library preparation following  shearing and purification.  All library preparation reaction volumes were scaled to accommodate input mass for a  given sample.  Library size selection was performed using the Sage Science BluePippin  0.75% Agarose, Dye Free, PacBio ~20kb templates, S1 cassette (PN# PAC20KB).  Size  selections were run overnight to maximize recovered mass. Approximately 2-5 mg of  prepared libraries were size selected using a 10 kb start and 50 kb end in ""Range"" mode.   This selection is necessary to narrow the library distribution and maximize the SMRTbell  sub-read length for the best de novo assembly possible.  Without selection, smaller  2000 - 10,000 bp molecules dominate the zero-mode waveguide loading distribution,  decreasing the sub-read length.  Size-selection was confirmed using pre and post size  selected DNA using an Agilent DNA 12000 chip. Final library mass was measured using the  Qubit High Sensitivity dsDNA Assay. Approximately 15-20% of the initial gDNA input mass  resulted after elution from the agarose cassette, which was enough yield to proceed to  primer annealing and DNA sequencing on the PacBio RSII instrument.  This entire library  preparation and selection strategy was conducted 7, 2 and 2 times across HG002, HG003,  and HG004 respectively, to provide enough library for the duration of this project.  Sequencing reflects the P6-C4 sequencing enzyme and chemistry, respectively. (Note that  10.3 % of the data was collected using the P5-C3 enzyme/chemistry prior to the release  of the P6-C4 enzyme and chemistry.)  Primer was annealed to the size-selected SMRTbell  with the full-length libraries (80ºC for 2 minute 30 followed by decreasing thetemperature  by 0.1º/s to 25Cº. To prepare the polymerase-template complex, the SMRTbell template complex  was then bound to the P6 enzyme using the Pacific Biosciences DNA Polymerase Binding Kit  P6 v2 (PN# 100-372-700). A ratio of 10:1, polymerase to SMRTbell at 0.5 nM, was prepared  and incubated for 4 hours at 30ºC and then held at 4ºC until ready for magbead loading  prior to sequencing.  The Magnetic bead-loading step was conducted using the Pacific  Biosciences MagBead Kit (PN# 100-133-600) at 4ºC for 60-minutes permanufacturer's guidelines.   The magbead-loaded, polymerase-bound, SMRTbell libraries were placed onto the RSII instrument at a sequencing concentration of 100 to 40 pM to optimize loading across various SMRTcells. Sequencing was performed using the C4 chemistry provided in the Pacific Biosciences DNA  Sequence Bundle 4.0 (PN# 100-356-400).  The RSII was then configured for 240-minute  continuous sequencing runs. "