Accel ngs 2s hyb dna library kit

The FFPE sections of tumor tissues that were obtained from the enrolled patients were pathologically confirmed by two independent pathologists to ensure their accurate diagnosis. All of the samples were carefully selected to have a tumor cell content exceeding 20%. DNA from FFPE samples was extracted by using the DNeasy Blood & Tissue Kit (Qiagen, Inc.) according to the manufacturer's instructions. The purified DNA was quantified by using a Qubit 3.0 fluorometer (Life Technologies, Inc.) with the following qualifying criteria: extraction concentration greater than 0.3 ng/μl and a total amount greater than 10 ng. The quality of DNA was assessed by using a PCR assay on the StepOnePlus System (Life Technologies, Inc.), with the QC criterion being a Qubit/qPCR value greater than 15. All of the collected samples successfully passed the quality control (QC) step. The Accel-NGS 2S HYB DNA LIBRARY KIT (Swift Biosciences, 23,096) and HotStart ReadyMix (KAPA, KK2612) were used for library preparation and amplification, respectively. The amplified libraries were purified by using SPRI SELECT (Beckman, B23319). Somatic genomic alterations were identified from the tumor samples by excluding the germline alterations that were identified in the matched blood samples.

First, more than 10 ng of genomic DNA was broken into an average fragment size of 300 base pairs by using the Covaris ultrasonic disruptor (E210, Covaris Inc., Massachusetts, USA). Thereafter, the sequencing libraries were prepared with the Accel-NGS® 2S Hyb DNA Library Kit (Swift Biosciences, California, USA), including end repair, base addition, and adaptor ligation steps. KAPA HiFi HotStart ReadyMix PCR Kit (Kapa Biosystems, Boston, USA) was adopted for the library enrichment. The PCR-amplified DNA-seq library quality was thereafter assessed using the Agilent DNA 1000 kit (Agilent Technologies, California, USA) on the Agilent 2100 BioAnalyzer (Agilent Technologies, California, USA) and quantified by using Qubit 3.0 Fluorometer (Life Technologies, California, USA).

Library preparation was performed as previously described [28 ] using either the Accel-NGS 2S Hyb DNA Library Kit (SWIFT Biosciences, San Francisco, CA, USA) or Twist Library Preparation EF Kit (TWIST Biosciences, San Francisco, CA, USA) [28 ]. For exome capture, either the Twist Human Core Exome Kit (TWIST Biosciences, San Francisco, CA, USA) or Clinical Research Exome V2 (Agilent, Santa Clara, CA, USA) were used and further sequenced by Illumina paired-end sequencing producing a minimum of 26 Gb and 18 Gb of raw sequence data for tumor DNA and normal DNA samples, respectively.