Formalin-fixed paraffin-embedded (FFPE) tissues was collected voluntary at baseline, DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Peripheral blood samples were collected at baseline and C1D31 respectively, plasma and white blood cells DNA were extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) and the Hipure Blood&Tissue DNA Kit (Magen Biotechnology, Guangzhou, China). The DNA obtained was captured by SureSelect XT-HS Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA) using a 212-gene panel (Table S1 , Repugene Technology, Hangzhou, China) for all samples. The constructed libraries were sequenced with mean sequencing depths approximately 20,000 times for blood samples and 4,000 times for tissue samples using the Illumina HiSeq-X10 platform (Illumina, San Diego, CA, USA). Then, the samples and white blood cells were paired to filter out clonal hematopoiesis variants and germline mutations. MuTect2 was used for single-nucleotide variation and insert-deletion variant detection and LUMPY (version 0.2.13) was used for gene fusion detection (16 (link)). The copy number variation was detected using the CNVkit software (v0.9.5). Mutation load of each patient was the sum of the number of detected mutation and ALK mutation abundance was defined as the variant allele fractions of ALK mutations.
Sureselect xt hs target enrichment system
Manufactured by Agilent Technologies
Sourced in United States
The SureSelect XT-HS Target Enrichment System is a laboratory equipment product from Agilent Technologies. It is designed for the enrichment and capture of specific genomic regions of interest prior to sequencing. The system utilizes hybridization-based target capture technology to selectively enrich DNA or RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 550dx platform, by Illumina (3 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions)
Hiseq x10 platform, by Illumina (1 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
Nextseq 550, by Illumina (1 mentions)
Nexseq 500, by Illumina (1 mentions)
Maxwell csc dna ffpe kit, by Promega (1 mentions)
Maxwell 16 ffpe tissue lev dna purification kit, by Promega (1 mentions)
Kapa hifi hotstart pcr kit, by Roche (1 mentions)
Kapa qpcr library quantification kit, by Roche (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
4200 tapestation system, by Agilent Technologies (1 mentions)
8 protocols using sureselect xt hs target enrichment system
1
Comprehensive Molecular Profiling of FFPE and Liquid Biopsies
2
Targeted DNA Sequencing of FFPE Tumors
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DNA was extracted from freshly cut slides from formalin fixed and paraffin embedded tumor blocks after manual microdissection of tumor areas using the Qiagen QIAamp DNA FFPE Tissue Kit. Libraries were prepared with a customized hybrid capture based approach using probes directed against a ~ 315 kbp region upstream of the NR4A3 TSS and the SureSelectXT HS Target Enrichment System (Agilent). After quality control, libraries were sequenced on NextSeq 550 (Illumina). Alignment and variant calling was done with the SureCall software for the customized hybrid capture approach (Agilent). Translocations were filtered by a quality score greater or equal to 255, and by a minimal support of the translocation of 15 reads. The remaining translocations were visually inspected using the Integrated Genomics Viewer (IGV) software58 (link),59 (link) and further filtered.
3
NGS Assay for Lung Cancer Profiling
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Lung Cancer Panel v3.0 is a laboratory-developed DNA- and RNA-based NGS assay to target 75 DNA genes (DNA) and 21 genes (RNA), respectively, including MET exon 14 skipping events along with prominent fusions. For the DNA-based assay, a library was prepared using the SureSelect XT-HS Target Enrichment System (Agilent Technologies) and the RNA-based assay utilized the anchored multiplex polymerase chain reaction method (ArcherDx, Boulder, CO) to enrich targets. Paired-end sequencing was performed on the Illumina NextSeq 550Dx platform (Illumina).
4
Targeted Sequencing of Breast Cancer Genes
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The gene panel was designed by SureDesign (https://earray.chem.agilent.com/suredesign ) to cover a whole exon of 13 genes including 12 frequently mutated genes (cBioPortal for cancer genomics, http://www.cbioportal.org ) in breast cancers and ESR1 (Supplementary Table 1). The median coverage of sequencing area was 100% (range, 74%-100%) of the whole exon in each gene. Tumor DNA was fragmented by Covaris S220 (Covaris, Inc., Woburn, MA, USA) to 150–200 bp, and 120 ng of them were used for sequencing. Sequence libraries were prepared with a custom SureSelect XT HS Target Enrichment System (Agilent Technologies, Inc. Santa Clara, CA, USA) according to the manufacturer's instructions and sequenced with Illumina MiSeq (Illumina, San Diego, CA, USA). SureCall ver4.0 (https://www.agilent.com/en/download-software-surecall ) was used for variant calling. The exclusion criteria were as follows; in introns, with VAF of <5%, with a depth of <300 reads, or reported in dbSNP/1000G database with a population of ≥1%. Variants obtained by panel sequencing were confirmed by Sanger sequencing using paired PBL DNA to exclude the patient-specific SNPs. Four hotspot mutations (AKT1-E17K and PIK3CA-E542K/E545K/H1047R) with VAF ≥1% were also included.
5
Targeted Deep Sequencing of Myeloid Malignancies
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Samples from 187 patients underwent targeted deep sequencing using an update of a previously validated in-house custom capture-enrichment panel [19 (link)] (SureSelect XT HS Target Enrichment System, Agilent Technologies, Santa Clara, CA, USA) of 92 genes related to the pathogenesis of myeloid malignancies (Table S1 ). Sequencing libraries were prepared according to manufacturer’s instructions and sequenced on Illumina MiSeq or NexSeq 500 sequencers.
The mean coverage of TDS was 665× (range 251–1198) and 99.5% of target regions were captured at a level higher than 100×.
For true oncogenic somatic variant calling, a severe criterion for variant filtering was applied. Variants were considered candidate somatic mutations based on the following criteria: (i) variants with ≥10 mutated reads; (ii) described in COSMIC and/or ClinVar as being cancer-associated and known hotspot mutations; and (iii) classified as deleterious and/or probably damaging by PolyPhen-2 and SIFT web-based platforms, as previously described [19 (link)].
The mean coverage of TDS was 665× (range 251–1198) and 99.5% of target regions were captured at a level higher than 100×.
For true oncogenic somatic variant calling, a severe criterion for variant filtering was applied. Variants were considered candidate somatic mutations based on the following criteria: (i) variants with ≥10 mutated reads; (ii) described in COSMIC and/or ClinVar as being cancer-associated and known hotspot mutations; and (iii) classified as deleterious and/or probably damaging by PolyPhen-2 and SIFT web-based platforms, as previously described [19 (link)].
6
Targeted NGS for Lymphoma Profiling
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Targeted NGS was performed for cases with available FFPE tissue samples using a customized panel of 121 lymphoma-related genes (SNUH FIRST-Lymphoma Panel v1.1; Supplementary Table S1, Supplemental Digital Content 2, http://links.lww.com/PAS/B366 ). ABCL01 had previous sequencing data, obtained from SNUH FIRST-Lymphoma Panel v1.0.
Genomic DNA was extracted from FFPE tissues using the Maxwell CSC DNA FFPE Kit or Maxwell 16 FFPE Tissue LEV DNA Purification Kit (Promega, Madison, WI). Libraries were developed using the SureSelect XT-HS Target Enrichment System (Agilent Technologies, Santa Clara, CA). Paired-end sequencing was performed using the NextSeq. 550Dx platform (Illumina Inc., San Diego, CA). Sequenced reads were aligned to the reference human genome (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA v0.7.17) and GATK Best Practice (v4.0.2.1). Single-nucleotide variants (SNVs) and small insertions and deletions (indels) were detected by using an in-house developed pipeline, SNVer (v0.5.3) and LoFreq (v2.1.2). Translocations were detected using Delly and Mantana, and copy number alterations were called using CNVkit. Mutations were annotated using SnpEff (v4.3).
Genomic DNA was extracted from FFPE tissues using the Maxwell CSC DNA FFPE Kit or Maxwell 16 FFPE Tissue LEV DNA Purification Kit (Promega, Madison, WI). Libraries were developed using the SureSelect XT-HS Target Enrichment System (Agilent Technologies, Santa Clara, CA). Paired-end sequencing was performed using the NextSeq. 550Dx platform (Illumina Inc., San Diego, CA). Sequenced reads were aligned to the reference human genome (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA v0.7.17) and GATK Best Practice (v4.0.2.1). Single-nucleotide variants (SNVs) and small insertions and deletions (indels) were detected by using an in-house developed pipeline, SNVer (v0.5.3) and LoFreq (v2.1.2). Translocations were detected using Delly and Mantana, and copy number alterations were called using CNVkit. Mutations were annotated using SnpEff (v4.3).
7
Comprehensive cfDNA Sequencing Protocol
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cfDNA and germline DNA from all CUP samples were processed in accordance with TARGET trial laboratory protocol50 (link) (TARGET patients up to TAR00286) or by the following updated method (TAR00287 onward and Biobank samples). DNA repair and dA-tailing were performed using the NEBNext® UltraTM II End Repair/dA-tailing Module (New England Biolabs, catalogue number E7546L). Adapter ligation and indexing was carried out using KAPA HyperPrep Kit (Roche, catalogue number 07962355001) with NEBNext® Multiplex Oligos for Illumina (New England Biolabs, catalogue number E7335L). Targeted NGS for whole genome libraries from cfDNA and corresponding germline DNA was carried out using SureSelect Custom DNA Target Enrichment Probes (Agilent, catalogue number 5190-4822). Target enrichment of 0.5–1.0 µg of each DNA library (paired cfDNA and gDNA libraries from the same patient pooled per pull down) was performed using SureSelect XT HS Target Enrichment System (Agilent, catalogue number G9703A) with a 641 gene hybridisation panel. Captured libraries were amplified using KAPA HiFi HotStart PCR Kits (Roche, catalogue number 07958897001) and quantified using the KAPA Library Quantification qPCR Kit (Roche, catalogue number 07960140001). Libraries were paired end sequenced at 2 ×150 bps on a NextSeq 500 or NovaSeq 6000 (Illumina).
8
Gene Panel for Lymphoid Neoplasm
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We created a customized panel composed of 120 genes important in the pathogenesis of lymphoid neoplasm (Supplementary Table S2, Supplementary Digital Content 3, http://links.lww.com/PAS/B300 ). At least 50 ng genomic DNA was extracted from each FFPE sample using the Maxwell FFPE Purification Kit (Promega, Madison, WI). Library preparation was performed using the SureSelect XT-HS Target Enrichment System (Agilent Technologies, Santa Clara, CA). Library concentrations were quantified and assessed by the 4200 TapeStation System (Agilent Technologies). Paired-end sequencing was performed on the Illumina NextSeq. 550Dx Platform (Illumina Inc., San Diego, CA).
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