Genetic analysis was conducted as previously described (25 (link), 26 (link)). Briefly, ctDNA was isolated from 4 to 5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA). Serial sections from formalin-fixed paraffin-embedded tumor tissues were used for genomic tumor DNA extraction using the QIAamp DNA mini kit (Qiagen, Valencia, CA). DNA from leukocytes was extracted using the DNeasy Blood Kit (Qiagen, Valencia, CA). Sequencing libraries were prepared from ctDNA using KAPA DNA Library Preparation Kits (Kapa Biosystems, Wilmington, MA, USA), and genomic DNA sequencing libraries were prepared with Illumina TruSeq DNA Library Preparation Kits (Illumina, San Diego, CA). Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Roche NimbleGen, Madison, WI, USA) targeting 1,021 genes (Supplementary Table S1 ), including MLH1, MSH2, MSH6, and PMS2. Prepared libraries were sequenced on a NextSeq CN 500 (Illumina, San Diego, CA).
Nextseq cn500
Manufactured by Illumina
Sourced in United States
The NextSeq CN500 is a next-generation sequencing (NGS) system designed for clinical research applications. It utilizes Illumina's sequencing-by-synthesis technology to generate high-quality sequencing data. The system is capable of processing a wide range of sample types and can be used for a variety of applications, including targeted gene sequencing, exome sequencing, and RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Kapa dna library preparation kit, by Roche (3 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Dneasy blood kit, by Qiagen (2 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (2 mentions)
Qubit dsdna hs high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Truseq dna library preparation kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Dna 1000 kit, by Agilent Technologies (1 mentions)
Nimblegen seqcap hybridization and wash kit, by Roche (1 mentions)
Qiaamp viral rna kit, by Qiagen (1 mentions)
Nextera xt dna library prep kit, by Illumina (1 mentions)
Qiaamp ucp pathogen dna kit, by Qiagen (1 mentions)
Ki 67, by Maixin Group (1 mentions)
Ttf 1, by Maixin Group (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Hypercap target enrichment kit, by Roche (1 mentions)
21 protocols using nextseq cn500
1
Comprehensive Genomic Profiling of ctDNA
2
Genome-wide CNV Detection by Sequencing
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Single end sequencing was performed on the NextSeq CN500 platform (Illumina, San Diego, CA, USA) with a run time of 6.5 h to generate approximately 5 million raw 45 bp reads per sample. Raw reads were then edited to remove artificial adaptor sequences, and the true 36 bp genome sequences were then mapped to the hg19 reference genome using the Burrows and Wheeler algorithm [18 (link)]. On average, approximately 2.8–3.2 million reads were uniquely mapped for data analysis. Reads were allocated to 20 kb bins along the length of each chromosome, and CNVs were identified from 24 chromosome copy number (CN) plots, as previously described [13 (link),19 (link)]. Duplications were defined as CN >2.8, deletions CN <1.2, disomy (1.8 < CN < 2.2), mosaic trisomy (2.2 < CN < 2.8) and mosaic monosomy (1.2 < CN < 1.8).
Genomic variant databases including DGV (Database of Genomic Variants),http://projects.tcag.ca/variation ), OMIM (Online Mendelian Inheritance in Man) (http://www.omim.org ), PubMed (http://www.ncbi.nlm.nih.gov/pubmed ) and UCSC (University of California, Santa Cruz) (http://genome.ucsc.edu/ , hg19) were used as a reference source of CNVs. The pathogenicity of detected CNVs was assessed following ACMG guidelines [20 (link)].
Genomic variant databases including DGV (Database of Genomic Variants),
3
Targeted Cancer Gene Panel Sequencing
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Targeted region selection was performed with the NimbleGen SeqCap Hybridization and Wash Kit (Roche, Swiss). A 1-µg mixed DNA library from 8-12 indexed Illumina libraries was captured with a hybridization probe. The probe library was designed through the NimbleDesign portal (Version 02) using genome build hg19 NCBI Build 37.1/GRCh37. The panel was 40 kb, targeting the regions of genes KRAS, NRAS, PIK3CA, BRAF, EGFR, EML4, ALK, SLC34A2, and ROS1. The hybridization and washing were conducted according to the manufacturer's protocol, and the captured DNA fragments were divided into two 50 µL reactions and amplified with 14 PCR cycles. The two reactions were pooled and purified with Agencourt AMPure XP beads. The captured product was sequenced using 150 bp paired-end runs on the Illumina NextSeq CN500 after quantification by the Qubit dsDNA Assay Kit and determination of fragment length by the Agilent 2100 Bioanalyzer with the DNA 1000 Kit. The median yield of each library was ~11.0 M reads, and the mean depth was ~10,000 X.
4
Comprehensive Plasma Nucleic Acid Extraction and Sequencing
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The nucleic acid extraction, library preparation and sequencing were carried out as previous study.17 (link) Briefly, approximately 3–5 mL peripheral blood was collected, and plasma was obtained by a centrifugation at 4 °C, 1600 rpm, for 10 min. Plasma DNA and RNA were extracted by using QIAamp® UCP Pathogen DNA Kit and QIAamp® Viral RNA Kit (Qiagen) according to the manufacturer’s instructions, respectively. To include negative controls, PBMC samples (105 cells/mL) obtained from healthy donors were prepared and underwent the same protocol and processing conditions as the test sample materials. Additionally, sterile deionized water was included as a non-template control (NTC) alongside the specimens. Finally, to help to differentiate pathogenic microorganisms from background microorganisms, a comprehensive list of suspected background or contaminated microorganisms were included at the end of the reports. Nextera XT DNA Library Prep Kit (Illumina) was used to construct libraries for sequencing (Illumina Nextseq CN500).
5
NSCLC Histopathology and EGFR Mutation
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The histopathological types and malignant degrees of NSCLCs were judged by experienced pathologists according to the immunohistochemical markers. During immunohistochemical analysis, pathologists judged the pathological types according to the following immune markers: TTF-1, NAP-A, CK, CK, Ki67 (Maixin, Fuzhou, China), which were used to classify the tumors into adenocarcinoma, SCC, adenosquamous carcinoma, large cell carcinoma, and other rare types.
Tumor EGFR mutations were detected by fiberoptic bronchoscopy, percutaneous lung puncture, or metastatic lymph node biopsy. In the absence of the above tissues, EGFR mutations can also be detected using the cytological samples of serous effusion. PCR (Next Seq CN500, Illumina, USA) was used to analyze the existence/absence of EGFR gene mutation. If no exon mutation was detected, the mutation was identified as “EGFR wild type,” whereas if any exon mutation is detected, it was identified as “EGFR mutation.”
Tumor EGFR mutations were detected by fiberoptic bronchoscopy, percutaneous lung puncture, or metastatic lymph node biopsy. In the absence of the above tissues, EGFR mutations can also be detected using the cytological samples of serous effusion. PCR (Next Seq CN500, Illumina, USA) was used to analyze the existence/absence of EGFR gene mutation. If no exon mutation was detected, the mutation was identified as “EGFR wild type,” whereas if any exon mutation is detected, it was identified as “EGFR mutation.”
6
cfDNA Library Preparation for Sequencing
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cfBEST tag adaptors were ligated to cfDNA after A-tailing was added to cfDNA using the KAPA Hyper Prep Kit (Kapa Biosystems, Boston, USA). The pre-library amplified from cfDNA with an index primer and a universal primer was split into two parts [named “F” and “R”, Additional file 2 : Fig. S1 shows only the “F” part)] to be amplified separately with two rounds of PCR. The two PCR products were pooled to execute a third PCR with universal primers U1 and U2 (Additional file 2 : Fig. S1). Finally, the sequencing libraries from the three rounds of PCR were subjected to massively parallel sequencing on the NextSeq CN500 (Illumina, San Diego, USA).
7
Targeted Capture and Sequencing of Circulating Cell-Free DNA
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DNA concentration was measured using the Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and the Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen, Carlsbad, CA, USA). The size distribution of circulating cfDNA was assessed using the Agilent 2100 BioAnalyzer and DNA HS kit (Agilent Technologies, Santa Clara, CA, USA). The SeqCap EZ Library system (Roche NimbleGen, Madison, WI, USA) was used for target enrichment. In total, 1,386 libraries from 970 patients were hybridized to custom-designed biotinylated oligonucleotide probes (IDT, Coralville, IA, USA) covering 1.6 Mbp of the genome, and the captured genomic regions included 1,021 cancer-related genes (Table S1
8
Library Preparation and Sequencing for Tissue and Plasma Samples
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In accordance with the manufacturer's instructions, the sequencing libraries were prepared using the KAPA DNA library preparation kit (Kapa Biosystems). Hybridization of barcoded libraries was performed on a previously reported customized 1021 panel (Table S1 ) for tissue samples and a 338 panel with ultra‐deep (Table S2 ) for plasma samples. After hybrid selection, DNA fragments were amplified and pooled into multiple multiplexed libraries. DNA sequencing was performed with the Illumina Nextseq CN 500 (Illumina) or the Gene+Seq‐2000 Sequencing System (GenePlus‐Suzhou).
9
Comprehensive Cancer Cell Sequencing
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We sent the patient biopsy puncture sample containing cancer cells for Next Generation Sequencing using Illumina NextSeq CN 500 (Shanghai Da An medical Laboratory, third-party business services). Average coverage depth of the sequencing was 8917x.
10
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 ).
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