For cancer patients, the libraries of genomic DNA and cfDNA were captured using an in‐house designed panel spanning a 1.89‐Mb genomic region and including 468 genes (Table S3 ). For asymptomatic individuals, the libraries of genomic DNA and cfDNA were captured using another in‐house‐designed panel spanning a 0.55‐Mb genomic region and including 118 genes (Table S4 ). Capture of the targeted regions was performed using a HyperCap Target Enrichment Kit (Roche). The hybridization and washing steps were conducted according to the manufacturer's protocol. Next, the captured libraries were sequenced using the NovaSeq 6000 system (Illumina) according to the manufacturer's protocol, producing paired‐end reads with a 150‐bp length at each end. For cancer patients, genomic DNA extracted from PBL was sequenced at a depth of at least 200×, while cfDNA extracted from plasma and genomic DNA extracted from FFPE tumour samples were sequenced at a depth of at least 1000×. For asymptomatic individuals, genomic DNA extracted from PBL and cfDNA extracted from plasma were sequenced with mean depths of 1723× and 2383×, respectively.
Hypercap target enrichment kit
Manufactured by Roche
Sourced in Switzerland
The HyperCap Target Enrichment Kit is a laboratory equipment product designed for targeted DNA sequencing. The kit includes reagents and protocols for selective capture and enrichment of specific genomic regions of interest prior to next-generation sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (5 mentions)
Kapa hyper prep kit, by Roche (2 mentions)
Kapa hifi hotstart readymix, by Roche (2 mentions)
Nextseq cn500, by Illumina (1 mentions)
Kapa high throughput library preparation kit, by Roche (1 mentions)
Seqcap ez hypercap workflow, by Roche (1 mentions)
Kapa hyperexome probes, by Roche (1 mentions)
Quantus fluorometer with quantifluor dsdna system kit, by Promega (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Sequencing platform, by Illumina (1 mentions)
S2 focused ultrasonicator, by Covaris (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Universal dna library prep kit, by Vazyme (1 mentions)
Magpure ffpe dna kit b, by Magen Biotechnology Co (1 mentions)
8 protocols using hypercap target enrichment kit
1
Cancer and Asymptomatic Targeted Sequencing
2
Cancer Gene Capture and Sequencing
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A fraction of each library was hybridized to a predesigned panel covering 1.6 Megabase of genomic regions covering 543 cancer-related genes using HyperCap Target Enrichment Kit (Roche, 8286345001 Swiss). After hybridization and washing according to the manufacturer’s protocol, the KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Massachusetts, KK2602 USA) was used for the amplification of captured libraries. Then, the capture libraries were purified with 1X AMPure, quantified, and pooled for sequencing on Illumina Novaseq 6000 with Paired end 150 bp mode.
3
Comprehensive Cancer Gene Profiling
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Tumor tissue specimens and matched peripheral blood cells underwent next-generation sequencing targeting 543 or 769 cancer-related genes (including HLA-A/B/C) at a CAP-certified laboratory or in the Hospital. DNA libraries were captured by HyperCap Target Enrichment Kit (Roche) and sequenced on the instruments of Illumina Novaseq. 6000 or NextSeq CN500. The average deduped sequencing depths of tissues and blood cells were ×830 and ×240, respectively. More details of sequencing and data analyses pipeline were described in the Supplementary Methods (Supplemental Digital Content 3, http://links.lww.com/JS9/A234 ).
4
Targeted NGS of Microdissected Tumor Samples
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Tumor areas were microscopically selected on HE‐stained slides, and the marked region was isolated from serial 5‐µm‐thick unstained sections by microdissection to obtain a tumor cell content (>70%) as assessed microscopically prior to DNA extraction (except for the lymph node metastasis of patient OC122 with an uncertain fraction of tumor cells). The SeqCap EZ HyperCap Workflow (Roche) was used for targeted next‐generation sequencing (NGS). The Kapa High Throughput Library Preparation Kit (Roche) with 1 µg of sonicated gDNA with an average length between 180 and 220 bp (Covaris M220) was used for end repair and A‐tailing, adapter ligation, and library amplification. The HyperCap Target Enrichment Kit (Roche) was used for hybridizing the libraries, washing, and amplification of captured libraries. MiSeq Reagent v3 600‐cycle kit and a MiSeq were used for paired‐end sequencing (2 × 150 cycles).
5
Whole Exome Sequencing Protocol
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Whole exome sequencing was performed using the Illumina sequencing platform. We prepared a gDNA library with 500 ng of gDNA sheared to ∼300 bp using a Covaris S2 focused-ultrasonicator, according to the manufacturer’s protocols. Upfront DNA fragmentation and DNA library preparation were conducted using the KAPA HyperPrep Kit (Roche Applied Science, Pleasanton, CA, USA) according to the manufacturer’s protocol. Library quantitation was performed using a Quantus Fluorometer with QuantiFluor® dsDNA System kit (Promega, Madison, WI, USA). A High Sensitivity DNA assay with gel electrophoresis on the 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA) was used for DNA sizing accuracy and quality control (between 300 and 400 bp).
To generate exome-enriched DNA libraries, the HyperCap Target Enrichment kit and KAPA HyperExome Probes (Roche Applied Science, Pleasanton, CA, USA) were used according to the manufacturer’s protocol.
According to the Illumina protocol, paired-end reads of 2 × 100 bp were loaded on the HiSeq 2500 System for further sequencing.
To generate exome-enriched DNA libraries, the HyperCap Target Enrichment kit and KAPA HyperExome Probes (Roche Applied Science, Pleasanton, CA, USA) were used according to the manufacturer’s protocol.
According to the Illumina protocol, paired-end reads of 2 × 100 bp were loaded on the HiSeq 2500 System for further sequencing.
6
Targeted Region Enrichment for Illumina Sequencing
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For targeted region selection, we employed the HyperCap Target Enrichment Kit from Roche (Switzerland). Specifically, we utilized the SeqCap EZ MedExome Target Enrichment kit 384 Reaction panel, following the manufacturer’s instructions for hybridization and washing steps. Subsequently, the captured products were subjected to sequencing using the Illumina Novaseq 6000 instrument with a recommended 150 bp paired end run.
7
Comprehensive Genomic Profiling of FFPE Tumors
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Detail methods of DNA sequencing was performed as our previous study [12 (link)]. DNA panel was 2,189 kb in length, which covered full coding regions or hotspot mutation regions in 769 genes. The genomic DNA from formalin-fixed paraffin-embedded (FFPE) tumor samples was extracted using the MagPure FFPE DNA Kit B (Magen, China), fragmented into DNA pieces of approximately 200 bp using an enzymatic method (5 × FEA Enzyme Mix; Qiagen, CN), subsequently constructed DNA libraries using a VAHTS Universal DNA Library Prep Kit (Vazyme, China). The libraries of genomic DNA were captured with a 769 gene panel (Table S1) and sequenced using a HyperCap Target Enrichment Kit (Roche, Switzerland) and the NovaSeq 6000 system (Illumina, USA) according to the manufacturer’s protocols, respectively.
8
Targeted Cancer Sequencing Panel Development
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Targeted sequencing with a customized panel of 769 cancer-related genes was performed for somatic mutation identification38 (link). Briefly, between 30 and 300 ng of fragmented tumor tissue or WBC genomic DNA or between 10 and 50 ng of cfDNA was used for library construction with KAPA Hyper Prep Kit (Roche, Catalog# KK8504). Targeted regions were captured with HyperCap Target Enrichment Kit (Roche) according to manufacturer instructions. The enriched libraries were amplified with KAPA HiFi HotStart ReadyMix (Roche, Catalog# KK2602) and sequenced on Illumina Novaseq 6000 in 150-bp paired-end mode.
To determine the detection sensitivity of a classifier at 99% specificity, a logistic regression was estimated between classifier predictions versus log10(mean VAF) for 65 cancer plasma samples with corresponding tumor tissue and WBC samples. Clinical LOD was defined as the mean VAF for a cancer detection probability of 50%. The 95% confidence interval was estimated using a Gaussian approximation for the standard error of the logistic regression slope.
To determine the detection sensitivity of a classifier at 99% specificity, a logistic regression was estimated between classifier predictions versus log10(mean VAF) for 65 cancer plasma samples with corresponding tumor tissue and WBC samples. Clinical LOD was defined as the mean VAF for a cancer detection probability of 50%. The 95% confidence interval was estimated using a Gaussian approximation for the standard error of the logistic regression slope.
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