WGS analysis of normal liver sample (T00589)
WGS analysis of normal liver sample (T00589) using Illumina HiSeq 2500
T00589
Input genomic DNA (gDNA) for Synthetic Long Read (SLR) library construction was first quantified by Qubit Fluorometer High Sensitivity assay kit (Invitrogen, Carlsbad, CA, USA). gDNA quality was then assessed by agarose gel electrophoresis prior to library construction to ensure that gDNA was intact and of high molecular weight.  500ng of gDNA was diluted to 10ng/µL and sheared using Covaris g-Tube, by centrifuging at 6700 rpm for 1 minute, Eppendorf model 5424.  Reagents and instructions for SLR library construction were provided in Illumina's TruSeq SLR library prep kit, FC-126-1001. End repair, 3' A-tailing and adapter ligation were purified by bead-based separation in between each step.  8-10kb size DNA fragments were size-selected using a precast 0.8% Clonewell E-gel (Life Technologies, cat# G661808).  DNA fragments were extracted using Qiagen's QIAquick Gel Extraction Kit (Cat#28704). DNA fragments were then visualized using an Agilent DNA 1000 series II assay (Agilent, Santa Clara CA, USA) to ensure that size selection was successful.  qPCR was performed to ensure appropriate amount of adapter ligated fragments were added into the Long Range PCR and subsequent Tagmentation process.  Long Range PCR was carried out to enrich for fragments with adapters ligated on both ends followed by Tagmentation as per standard protocol.  384 indexes were introduced by Indexing PCR and the resulting PCR products were pooled, purified and concentrated.  Bead based size selection was performed to enrich for appropriately sized DNA fragments.  Constructed library fragments were assessed on Agilent HS DNA series II assay for size (bp), and concentration was measured by qPCR using Kappa Library KAPA Library Quantification Kit.