Library preparation was performed on genomic DNA samples using a panel of 94 cancer susceptibility genes (Illumina TruSight Cancer Sequencing panel—#FC-121-0202). The panel contains oligos targeting and enriching more than 1700 exons including coding regions and noncoding exon-flanking regions (~ 50 bp) spanning 94 cancer susceptibility genes (Additional file 1 ) [43 ]. The TruSight Rapid Capture kit was used for the library preparation according to the manufacturer’s protocol (Illumina, #FC-140-1106). Paired-end sequencing was performed on the NextSeq 500 Sequencing Platform (Illumina) using a High-Output v2.5 kit. We carried out two independent runs of 2 × 75 cycles and 2 × 150 cycles, aiming to examine the effect of read depth on subsequent variant calls.
Trusight cancer sequencing panel
Manufactured by Illumina
Sourced in United States, United Kingdom
The TruSight Cancer sequencing panel is a laboratory equipment product designed to analyze a comprehensive set of genes associated with a variety of cancer types. It provides a targeted approach to DNA sequencing, focusing on specific genomic regions of interest related to cancer. The core function of this product is to enable researchers and clinicians to detect and investigate genetic variations that may be linked to cancer development and progression.
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Lab products found in correlation
Miseq platform, by Illumina (4 mentions)
Dneasy tissue kit, by Qiagen (4 mentions)
Trusight rapid capture kit, by Illumina (3 mentions)
Nextseq system, by Illumina (3 mentions)
Trusight rapid capture, by Illumina (2 mentions)
Miseq sequencer, by Illumina (2 mentions)
Misequ, by Illumina (2 mentions)
Nextseq 500 sequencing platform, by Illumina (1 mentions)
Varseq software, by Golden Helix (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Coffalyzer software, by MRC-Holland (1 mentions)
Mlpa p087, by MRC-Holland (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Qubit double stranded dna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Trurisk, by Agilent Technologies (1 mentions)
Haloplex, by Agilent Technologies (1 mentions)
24 protocols using trusight cancer sequencing panel
1
Targeted Cancer Gene Sequencing Protocol
2
Comprehensive Genomic Profiling Protocol
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Genomic DNA from peripheral blood sample or saliva was extracted and was used to perform capture by hybridization of the exons and exon-intron boundaries of the 94 genes using the commercial kits TruSight Cancer Sequencing Panel and TruSight Rapid Capture (Illumina). Next generation sequencing (NGS) was performed on the NextSeq 500 System (Illumina) platform. Sequences corresponding to the requested genes of each patient were compared with the respective reference sequences for calling variants using bioinformatics tools (Isaac Enrichment v3.0 and Illumina Variant Studio 2.2).
All identified variants were imported into the VarSeq software (Golden Helix) for function, classification and frequency annotations in public databases. Variants were filtered according to the criteria: quality >30; variant base present in at least 25% of the reads; absent in population databases (gnomAD, dbSNP, 1000genomes and Abraom - database of variants of exomes of the Brazilian population:http://abraom.ib.usp.br/ ) or, when present, presenting minor allele frequencies (MAF) ≤ 0.01.
Multiplex Ligation-dependent Probe Amplification (MLPA – P087, MRC-Holland, Amsterdam, NL) was used for BRCA1 and BRCA2 copy number variation analysis, according to the manufacturer’s recommendations. Coffalyzer software (MRC-Holland, Amsterdam, NL) was used at default settings for data analyses.
All identified variants were imported into the VarSeq software (Golden Helix) for function, classification and frequency annotations in public databases. Variants were filtered according to the criteria: quality >30; variant base present in at least 25% of the reads; absent in population databases (gnomAD, dbSNP, 1000genomes and Abraom - database of variants of exomes of the Brazilian population:
Multiplex Ligation-dependent Probe Amplification (MLPA – P087, MRC-Holland, Amsterdam, NL) was used for BRCA1 and BRCA2 copy number variation analysis, according to the manufacturer’s recommendations. Coffalyzer software (MRC-Holland, Amsterdam, NL) was used at default settings for data analyses.
3
Genetic Screening for Hereditary Gastric Cancer
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Patients with a family history of DGC and/or LBC but without prior diagnosis of a PV in CDH1 or CTNNA1 received genetic counseling. In patients fulfilling the 2015 HDGC testing criteria, Sanger sequencing of the CDH1 gene including the intron–exon boundaries or next generation sequencing based multigene panel (customized version of the TruSight™Cancer Sequencing Panel including CTNNA1; Illumina, San Diego, CA, USA) was performed using leucocyte DNA. Multiplex ligation-dependent probe amplification (MLPA) was used to detect large genomic rearrangements (MRC Holland, Amsterdam, The Netherlands). All patients presenting after August 2018 received sequencing of the CTNNA1 gene, in case no CDH1 PV had been detected. Interpretation of variants was based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines [54 (link)]. All variants were later assessed according to the current ACMG-AMP classification of CDH1 variants [33 (link)].
4
Targeted DNA Sequencing for Cancer Risk
5
Targeted Capture and Sequencing of Genomic DNA
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Genomic DNA libraries were prepared from snap frozen and SCP derived DNA using Illumina® TruSight Rapid Capture kit (Cat. No. FC-140-1101). According to the manufacturer’s protocol, 10 μL of DNA at 5 ng/10 μL (n = 3) was used in the tagmentation reaction, followed by limited-cycle PCR amplification. After clean up, the DNA libraries were pooled and hybridized with capture probes using Illumina TruSight Cancer sequencing panel (Cat. No. FC-121-0202). To ensure high specificity of the captured regions, a second hybridization and capture process was performed. The captured libraries were finally amplified via another limited-cycle PCR program and sequenced on an Illumina HiSeq 2500 sequence analyzer. The reads were pre-processed using fqtrim (http://ccb.jhu.edu/software/fqtrim/index.shtml ) to remove any N that was present. The resulting reads were sequentially aligned to hg19 human genome (UCSC version, February 2009) using the Burrows-Wheeler alignment tool.
6
NGS-based Cancer Gene Sequencing
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Next-generation sequencing (NGS) was performed using TruSight Rapid Capture Kit (Illumina, USA) in combination with the TruSight Cancer Sequencing Panel (Illumina, USA) according to the manufacturer's recommendations.
DNA libraries with 4 mM molarities were subjected to clustering using a standard flow cell and were sequenced on the MiSeq platform (Illumina) using the MiSeq Reagent Kit v3 (600 cycles).
DNA libraries with 4 mM molarities were subjected to clustering using a standard flow cell and were sequenced on the MiSeq platform (Illumina) using the MiSeq Reagent Kit v3 (600 cycles).
7
Comprehensive DNA Extraction and Sequencing Workflow
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Total DNA was extracted by using the NucleoSpin Tissue kit (Macherey Nagel). DNA concentration was determined using a Qubit double-stranded DNA High Sensitivity Assay Kit and Qubit fluorometer (ThermoFisher Scientific, Waltman, MA, USA). Secondary mutations in the KIT gene were determined using the TruSight™ Cancer Sequencing Panel (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Captured libraries were assessed for quality using the Agilent High Sensitivity DNA assay and yield using the Qubit double-stranded DNA High Sensitivity Assay Kit and the Qubit fluorometer (ThermoFisher Scientific, Whatman, MA, USA). The resulting library was sequenced using the Illumina MiSeq platform and MiSeq Reagent Kit v 2 (300 cycles) to generate 149-bp paired-end reads (2 × 149 PE). (“Illumina”) following the manufacturer’s protocol. Raw-data reads were aligned to the human reference genome (hg 38) using aligner BWA-MEM with BamQC, FastQC and NGSrich quality control checks. Single nucleotide polymorphism (SNP) analysis was performed using CLC Medical Workbench 3.0.
8
Multi-gene panel testing for non-BRCA1/2 cancer risk
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Genomic DNA was isolated from venous blood samples. NGS and data analysis were carried out at each participating center using Illumina sequencing platforms, employing either the customized TruRisk® (Agilent or Illumina), a customized HaloPlex (Agilent, Santa Clara, California, USA), or the TruSight™ Cancer Sequencing Panel (Illumina, San Diego, California, USA) for target enrichment. All gene panels covered the eight selected non‐BRCA1/2 core genes. The diagnostic pipelines of the labs involved have been successfully tested in European Molecular Genetics Quality Network (EMQN) schemes. Since LGRs cannot be detected reliably on the basis of NGS‐data 29 , this mutation type was not included in this study. All deleterious variants affecting canonical non‐BRCA1/2 core gene transcripts (ATM, NM_000051.3; CDH1, NM_004360.3; CHEK2, NM_007194; NBN, NM_002485.4; PALB2, NM_024675.3; RAD51C, NM_058216.2; RAD51D, NM_002878.3; TP53, NM_000546.5) were routinely verified by Sanger sequencing.
9
Targeted NGS for Cancer Predisposition
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Targeted NGS was performed on patient genomic DNA using the Trusight Cancer sequencing panel (Illumina) that contains 1,736 genomic regions from 94 genes suspected of having a role in cancer predisposition, including the RB1 gene. An analytical validation of our panel has shown a sensitivity of 98.2%, specificity of 100%, and reproducibility of 99.5%. The gene coverage analysis on this panel revealed that exonic and flanking intronic regions of the RB1 gene (NM_000321 ) showed coverage of >99% (≥ 20 reads) with a mean read depth of 405X. The Nextera DNA library preparation protocol (Illumina) to convert input genomic DNA (gDNA) into adaptor-tagged indexed libraries was essentially performed as previously described [15 (link)]. The tagged and amplified sample libraries were checked for quality and they were quantified using the BioAnalyzer (Agilent). Up to 6–10 pM of the pooled library was loaded and sequenced on the MiSeq platform (Illumina), according to the manufacturer’s instructions.
10
Targeted Next-Generation Sequencing for Cancer
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Genomic DNA was extracted from peripheral EDTA-anticoagulated blood samples using the standard salting-out procedure. NGS targeted mutation screening was performed using the TruSight™Cancer Sequencing Panel (Illumina, San Diego). This customized commercial kit includes 94 well established genes for HTS. The panel was extended using the Illumina DesignStudio by the addition of a further 54 relevant HTS genes (Additional file 3 : Table S2), based on literature search. In total, 2426 selected targets were sequenced based on 3939 probes, corresponding to a cumulative target length of 626,960 bp. Library preparation, target enrichment, and high-throughput sequencing were performed according to the manufacturer’s protocol. All samples were sequenced on an Illumina MiSeq sequencer. Alignment was perfomed using the software of the Illumina MiSeq sequencer or the SeqPilot software (JSI Medical Systems), based on hg19.
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