title: NA12878 P5-C3 Sequencing
design: NA12878 genomic DNA library preparation was prepared using high molecular weight DNA (20-50kb) extracted from the Coriell control sample and sequencing was performed using a modified method, primarily based on the manufacturer’s instructions.  GM12878 cells at passage 4 were obtained from Coriell. They were permitted two passages to insure recovery from transfer, then were expanded over an additional 4 passages, all in RPMI + 15% FBS without antibiotics. At this point 10^8 cells were spun down and genomic DNA was purified using eight QiaAMP DNA Mini columns.  DNA was verified using high sensitivity Qubit analysis to quantify the mass of double-stranded DNA present.  After quantification, DNA was diluted to 150 µL using Qiagen elution buffer at 33 µg / µL.  The 150 µL aliquot was individually pipetted into the top chamber of a Covaris G-tube spin column and sheared gently for 60 seconds at 4500 rpm using an Eppendorf 5424 bench top centrifuge.  Once completed, the spin column was flipped after verifying that all DNA was now in the lower chamber.  Then, the column was spun for another 60 seconds at 4500 rpm to further shear the DNA and place the aliquot back into the upper chamber, resulting in a 10,000 to 20,000 bp DNA shear, verified using a DNA 12000 Agilent Bioanalyzer gel chip.   The sheared DNA was then re-purified using a 0.45X AMPure XP purification step (0.45X AMPure beads added, by volume, to each DNA sample dissolved in 200 µL elution buffer (EB), vortexed for 10 minutes at 2,000 rpm, followed by two washes with 70% alcohol and finally diluted in EB). This AMPure XP purification step assures removal of any small fragment and/or biological contaminant.     After purification and shearing, ~1.6 to 3.2 ug of purified and sheared sample was taken into DNA damage and end-repair from each batch preparation.  Briefly, the DNA fragments were repaired using DNA damage repair solution (1X DNA damage repair buffer, 1X NAD+, 1 mM ATP high, 0.1 mM dNTP, and 1X DNA damage repair mix) with a volume of 21.1 µL and incubated at 37ºC for 20 minutes. DNA ends were repaired next by adding 1X end repair mix to the solution, which was incubated at 25ºC for 5 minutes, followed by the second 0.45X Ampure XP purification step. Next, 0.75 µM of blunt adapter was added to the DNA, followed by 1X template preparation buffer, 0.05 mM ATP low and 0.75 U/µL T4 ligase to ligate (final volume of 47.5 µL) the SMRTbell adapters to the DNA fragments. This solution was incubated at 25ºC overnight, followed by a 65ºC 10-minute ligase denaturation step. After ligation, the library was treated with an exonuclease cocktail to remove un-ligated DNA fragments using a solution of 1.81 U/µL Exo III 18 and 0.18 U/µL Exo VII, then incubated at 37ºC for 1 hour. Two additional 0.45X Ampure XP purifications steps were performed to remove < 2000 bp molecular weight DNA and organic contaminant.      Upon completion of library construction, samples were validated as ~20 kb using another Agilent DNA 12000 gel chip.  Blue Pippin size selection was deployed and the library was sufficient for additional size selection to remove any library molecules < 7,000 to 10,000 bp.  This step was conducted using Sage Science Blue Pippin 0.75% agarose cassettes to select library in the range of 7,000 bp – 50,000 bp.  This selection is necessary to narrow the library distribution and maximize the SMRTbell sub-readlength for the best de novo assembly possible. 14-26% of the input library resulted after elution from the agarose cassette, which was enough yield to proceed to primer annealing and DNA sequencing on the PacBio RSII machine. Size-selection was confirmed by Bio-Analysis and the mass was quantified using the aforementioned Qubit assay.
identifiers.namespace: PacBio
identifiers.id: P5-C3