Sureselect xt target enrichment kit

Targeted NGS was performed as described previously [13 (link)]. SureSelect-XT Target Enrichment Kit, https://www.agilent.com/en/product/next-generation-sequencing/hybridization-based-next-generation-sequencing-ngs/dna-seq-reagents-kits-library-preparation-kits/sureselectxt-reagent-kits-232859 (Agilent Technologies, Santa Clara, CA, United States) was used to prepare the DNA libraries. Briefly, 250 ng DNA was fragmented to a size of 250 to 300 bp, by a Covaris M220 sonicator. The DNA fragments were end-repaired and A-tailed, followed by adaptor ligation and subsequent amplification of the ligated DNA fragments using 10 cycles of polymerase chain reaction (PCR). Each amplified library was then hybridized to a SureSelect 640 genes oncogenesis custom panel 2.8 Mb bait set (Agilent Technologies, Santa Clara, CA, United States) according to the manufacturer's protocol (Individual gene list provided as supplementary material). Captured DNA was washed and amplified by 12 PCR cycles following the manufacturer's guidelines. Next, Tapestation 2200 (Agilent Technologies, Santa Clara, CA, United States) was used to assess the size and concentration of the captured DNA. Captured samples were pooled and sequenced on a single HiSeq flow cell on HiSeq 2500 (Illumina, San Diego, CA, United States), using a 2 × 100 bp PE Rapid Run v2 protocol.

Seven samples were used for Illumina sequencing. Library preparation was performed using Agilent SureSelectXT Target Enrichment kit according to the manufacturer’s instructions. Briefly, DNA was fragmented to 600 to 800 bp using micro-TUBE (Covaris). Fragments were end repaired, adapter ligated and amplified prior to target enrichment. Amplified fragments were hybridized to the designed capture probes for 24 h. After hybridization, Streptavidin beads were used to capture the DNA fragments bound to the probes. Captured DNA was amplified using Illumina indexing adapters. Amplified libraries were sequenced on Illumina MiSeq with 300 bp paired end sequencing. Samples NA12878 and NA12891 were sequenced as a pool of 4 samples in run 1 (other 2 samples in the pool are not included in the paper), whereas samples NA12877, NA12878, NA12879, NA12889, NA12890 and NA12892 were sequenced as a pool of 6 samples in run 2. Sample NA12878, which was used as reference sample was sequenced in both sequencing runs and data from sequencing run 1 was used as test sample and data from sequencing run 2 was used as reference sample.

DNA was extracted from fresh bone marrow aspirates and next-generation sequencing (NGS) testing was performed using a targeted NGS next-generation sequencing panel at each institution. The Mayo myeloid NGS panel includes 42 genes commonly mutated in MNs: ANKRD26, ASXL1, BCOR, CALR, CBL, CEBPA, CSF3R, DDX41, DNMT3A, ELANE, ETNK1, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, MPL, NPM1, NRAS, PHF6, PTPN11, RAD21, RUNX1, SETBP1, SH2B3, SF3B1, SRP72, SMC3, SRSF2, STAG2, TERT, TET2, TP53, U2AF1, WT1, and ZRSR2. The library preparation, sequencing and data analysis were described before (26 (link)). Briefly, libraries were prepared using the Agilent SureSelect‐XT Target Enrichment Kit (SureSelectXT, Agilent, Santa Clara, CA). and sequencing was performed on MiSeq or HiSeq platforms (Illumina, San Diego, CA) at the Mayo Clinic Clinical Genome Sequencing Laboratory. Different panels from other institutions were described in Supplementary Table 1, which are all clinical NGS assays performed in CLIA-CAP approved laboratories.

DNA was extracted from fresh bone marrow aspirates and next-generation sequencing (NGS) testing was performed using a targeted next-generation sequencing (NGS) panel that included 42 genes commonly mutated in myeloid neoplasms: ANKRD26, ASXL1, BCOR, CALR, CBL, CEBPA, CSF3R, DDX41, DNMT3A, ELANE, ETNK1, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, MPL, NPM1, NRAS, PHF6, PTPN11, RAD21, RUNX1, SETBP1, SH2B3, SF3B1, SRP72, SMC3, SRSF2, STAG2,TERT, TET2, TP53, U2AF1, WT1, and ZRSR2. The library preparation, sequencing, and data analysis were performed as described [15 (link)]. Briefly, libraries were prepared using the Agilent SureSelect‐XT Target Enrichment Kit (SureSelectXT, Agilent, Santa Clara, CA). and sequencing was performed on MiSeq or HiSeq platforms (Illumina, San Diego, CA) at the Mayo Clinic Clinical Genome Sequencing Laboratory. Pathogenic and likely pathogenic variants calling was performed as described [16 (link)]. Only the variants at the sites with a total read depth >100, supported by more than five alternate variant reads and a variant allele frequency (VAF) ≥ 5%, were retained for further analysis.

The Agilent SureSelect^XT target enrichment kit (cat no. G7530-90004) was used for bovine exome sequence enrichment. It covers a total of ~45 Mb of the bovine sequence that composed of the coding regions in the UMD3.1 reference genome (coding regions from Refseq and Ensembl, no UTR “untranslated regions”) and microRNA. The exomes of a further 10 KEASZ samples (Supplementary Table 1) were sequenced in the Deep Seq facility at the University of Nottingham using an ABI SOLiD 5500 genetic analyser. Using the LifeScope Enrichment Sequencing Pipeline, the generated 75 bp reads were mapped to the UMD3.1 bovine reference genome. Supplementary Table 2 summarizes the number of aligned reads, average depth of coverage, and percentage of reference exome for each sample. The same variants calling criteria used in the KEASZ whole genome sequence were implemented for the exome analysis except that reads with MAPQ ≥ 30 and base quality of 30 were used.