Tso 500 library preparation kit

DNA was isolated from BMA using the QIAamp DNA Blood Mini kit (QIAGEN, Hilden, Germany) as per the manufacturer’s protocol. Double-stranded DNA was measured using Qubit dsDNA broad-range assay kit (#Q32850, Invitrogen, Waltham, MA, USA) and 120 ng gDNA was used for library preparation. The libraries were prepared using the hybrid capture-based TSO 500 library preparation kit (#20028214, TruSight Oncology 500 DNA Kit, Illumina, San Diego, CA, USA) following the manufacturer’s instructions. In brief, the DNA was fragmented using an ultrasonicator (Covaris, Woburn, MA, USA) with a target peak of ~130 bp. After end repair, A-tailing, and adapter ligation, the adapter-ligated fragments were amplified using index PCR (UP-index) specific primers. Further, the libraries were enriched through a hybrid capture-based method using specific probes. This was followed by PCR-based enrichment, cleanup, and quantification of double-stranded DNA using high sensitivity Qubit (#Q32854 Invitrogen, Waltham, MA, USA) measurement. The libraries were subjected to bead-based normalization and were sequenced using V2 sequencing reagent kits on a NextSeq550 platform (Illumina, San Diego, CA, USA) as per manufacturer recommendations.

All samples passing quality control (QC) were subjected to library preparation using the hybrid capture-based TSO 500 library preparation kit (# 20028214, TruSight Oncology 500 DNA Kit, Illumina, San Diego, CA) following manufacturer’s instruction. In brief, the DNA was fragmented using an ultrasonicator (Covaris, Woburn, MA) with a target peak of ~130 bp. After end repair, A-tailing, and adapter ligation, the adapter ligated fragments were amplified using index PCR (UP-index) specific primers. Further, the libraries were enriched through hybrid capture based method using specific probes. This was followed by PCR based enrichment, cleanup, and quantification of double stranded DNA using high sensitivity Qubit (#Q32854 Invitrogen, USA) measurement. The libraries were subjected to bead based normalization and were sequenced using V2 sequencing reagent kits on a NextSeq550 platform (Illumina, San Diego, CA) as per manufacturer recommendations.

Genomic DNA and RNA samples were extracted from the same FFPE tissue sections using kit AllPrep DNA/RNA FFPE KIT (Cat No./ID:80234) according to the manufacturer’s protocols.
In this study, the preparation of both RNA and DNA sample library were performed according to the TSO500 Library Preparation Kit (Illumina, San Diego, CA, USA). Then, a two-step capture and enrichment of specific capture probes for DNA samples was performed, the DNA library and corresponding cDNA library of 8 samples was standardized by using the library homogenization method based on magnetic bead purification and sequenced using the Illumina NextSeq 550Dx platform. The Sequencing results were analyzed using TSO500 Docker pipeline.