title: WES of PPTC PDX models: Sample ETP-4
design: Illumina paired-end pre-capture libraries were constructed from PDX DNA samples according to the manufacturerâ€™s protocol (Illumina Multiplexing_SamplePrep_Guide_1005361_D) modified as described in the BCM-HGSC Illumina Barcoded Paired-End Capture Library Preparation protocol. The complete protocol including oligonucleotide sequences used as adaptors and blockers are accessible from the HGSC website https://www.hgsc.bcm.edu/sites/default/files/documents/Protocol-Illumina_Whole_Exome_Sequencing_Library_Preparation-KAPA_Version_BCM-HGSC_RD_03-20-2014.pdf. The DNA sequence production is described briefly below.Library Preparation. 500 ng (or 250 ng if sample quantity was limiting) of DNA in 50ul volume were sheared into fragments to an average size of 200-300 bp in a Covaris plate with E220 system (Covaris, Inc. Woburn, MA) followed by end-repair, A-tailing and ligation of the Illumina multiplexing PE adaptors. Pre-capture Ligation Mediated-PCR (LM-PCR) was performed for 6-8 cycles using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc.). Universal primer LM-PCR Primer 1.0 and LM-PCR Primer 2.0 were used to amplify the ligated products.  Reaction products were purified using 1.8X Agencourt AMPure XP beads (Beckman Coulter) after each enzymatic reaction.  Following the final 1.2X Agentcourt XP beads purification, quantification and size distribution of the pre-capture LM-PCR product was determined using Fragment Analyzer capillary electrophoresis system (Advanced Analytical Technologies, Inc.). Capture Enrichment. Four pre-capture libraries were pooled together (~750 ng/sample, 3 ug/pool) and then hybridized in solution to the HGSC VCRome 2.1 design1 (Bainbridge et al. 2011) according to the manufacturerâ€™s protocol NimbleGen SeqCap EZ Exome Library SR Userâ€™s Guide (Version 2.2) with minor revisions. Probes for exome coverage across >3,500 clinically relevant genes that are previously <20X (~2.72Mb) is supplemented into the VCRome 2.1 probe. Human COT1 DNA was added into the hybridization to block repetitive genomic sequences. Blocking oligonucleotides from Sigma (individually sequence specifically synthesized) or xGen Universal Blocking oligonucleotides (Integrated DNA Technologies) were added into the hybridization to block the adaptor sequences. Hybridization was carried out at 560C for ~16h. Post-capture LM-PCR amplification was performed using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc.) with 12 cycles of amplification. After the final AMPure XP bead purification, quantity and size of the capture library was analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500. The efficiency of the capture was evaluated by performing a qPCR-based quality check on the four standard NimbleGen internal controls. Successful enrichment of the capture libraries was estimated to range from a 6 to 9 of Î”CT value over the non-enriched samples. DNA Sequencing. Library templates were prepared for sequencing using Illuminaâ€™s cBot cluster generation system with TruSeq PE Cluster Generation Kits (Illumina) according to the manufacturerâ€™s protocol.  Briefly, these libraries were denatured with sodium hydroxide and diluted to 6-9 pM in hybridization buffer in order to achieve a load density of ~800K clusters/mm2. Each library pool was loaded in a single lane of a HiSeq flow cell, and each lane was spiked with 1% phiX control library for run quality control. The sample libraries then underwent bridge amplification to form clonal clusters, followed by hybridization with the sequencing primer. Sequencing runs were performed in paired-end mode using the Illumina HiSeq 2000 platform. Using the TruSeq SBS Kits (Illumina), sequencing-by-synthesis reactions were extended for 101 cycles from each end, with an additional 7 cycles for the index read. With sequencing yields averaging 12.1 Gb per sample, samples achieved an average of 97. 64% of the targeted exome bases covered to a depth of 20X or greater.
library_name: ETP-4-HUMAN-WES
attributes.tag: alignment_software
attributes.value: https://github.com/marislab/pdx-mouse-subtraction