Labchip gx electrophoresis system

Illumina libraries were constructed according to the manufacturer’s protocol with modifications as described in HGSC website (https://hgsc.bcm.edu/sites/default/files/documents/Illumina_Barcoded_Paired-End_Capture_Library_Preparation.pdf ). Libraries were prepared using Beckman robotic workstations (Biomek NXp and FXp models. Briefly, 1 ug of genomic DNA in 100ul volume was sheared into fragments of approximately 300–400 base pairs in a Covaris plate with E210 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 7 cycles of amplification using the 2X SOLiD Library High Fidelity Amplification Mix (a custom product manufactured by Invitrogen). Universal primer IMUX-P1.0 and a pre-capture barcoded primer IBC were used in the PCR amplification. In total, a set of 12 such barcoded primers were used on these samples. Purification was performed with Agencourt AMPure XP beads after enzymatic reactions. Following the final XP bead purification, quantification and size distribution of the pre-capture LM-PCR product was determined using the LabChip GX electrophoresis system (PerkinElmer).

DNA samples were constructed into Illumina paired-end pre-capture libraries according to the manufacturer’s protocol (Illumina Multiplexing_SamplePrep_Guide_1005361_D) with modifications as described in the BCM-HGSC protocol (https://hgsc.bcm.edu/sites/default/files/documents/Illumina_Barcoded_Paired-End_Capture_Library_Preparation.pdf). Libraries were prepared using Beckman robotic workstations (Biomek NXp and FXp models). Briefly, 1 ug of DNA was sheared into fragments of approximately 300–400 base pairs with the Covaris E210 system followed 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 of amplification using the 2X SOLiD Library High Fidelity Amplification Mix (a custom product manufactured by Invitrogen). Purification was performed with Agencourt AMPure XP beads after enzymatic reactions, and following the final purification, quantification and size distribution of the pre-capture LM-PCR product was determined using the LabChip GX electrophoresis system (PerkinElmer).