Xgen lockdown probes

Targeted Sequencing was performed with the illumina Hiseq Xten platform at the sequencing laboratory of Tissuebank Precision Medical Co, Ltd. (Shanghai, China). A total of 10 ng DNA per sample was amplified by PCR, and then the library was captured by using xGen^® Lockdown^® probes and xGen Hybridization and Wash Kit; Illumina Hiseq sequencer carried out pair end sequencing with a depth of 200X. 43 pathogenic genes (ASXL1, BCOR, BCORL1, BRAF, CALR, CBL, CDKN2A, CEBPA, CREBBP, CSF3R, CUX1, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, GNAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KIT, KRAS, MPL, NF1, NPM1, NRAS, PHF6, PIGA, PTEN, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2) were screened in all patients, including the entire coding regions and exon-intron boundaries. This multi-gene panel was expected to cover 100% of the targeted area. DNA was sheared into short genetic fragments (150∼200 bp) using the Covaris LE220, which included purified and captured gene fragments. Adaptor-ligated amplicons were prepared using the Illumina Paired-End Sample Preparation kit. Illumina multi-PE-adaptors were bound to terminal genes and target enrichment was performed by probe capture, amplicons were purified using VAHTS DNA Clean Beads and captured on the Illumina Hiseq Xten instrument.

For cell free DNA (cfDNA), library with unique molecular identifier tagging were prepared from 2 ng of cfDNA using the Accel-NGS 2S Plus DNA library kit (Swift Biosciences, USA) following the manufacturer’s instructions. Library concentrations were quantified with a QuantiFluor dsDNA system (Promega, USA). Equal amounts of libraries were pooled together and hybridized with xGen Lockdown probes for four targeted genes EGFR, KRAS, NRAS, BRAF (IDT DNA, USA). Sequencing was performed using NextSeq 500/550 High output kits v2 (150 cycles) on Illumina NextSeq 550 system (Illumina, USA) with the coverage of 10.000X.
For genomic DNA from FFPE, libraries were prepared from 2 ng of cfDNA using the NEBNext Ultra II FS DNA library prep kit (New England Biolabs, USA) following the manufacturer’s instructions. Similar to ctDNA libraries, FFPE libraries were pooled before hybridization with the xGen Lockdown probes and sequencing in the Illumina NextSeq 550 system. Both cfDNA and FFPE samples exhibited 5–10% on-target reads.