Base calls were generated on-instrument. Secondary data analysis was performed by the MiSeq Reporter 2.6.2.3 PCR Amplicon Workflow and aligned to human genome build 37. All samples were required to pass a quality control step of 250x coverage across 80% of the amplicon to be included in subsequent analyses. Variants were detected using the Illumina Somatic Variant Caller, designed to detect low- frequency mutations below 5% (Illumina Technical Note– Somatic Variant Caller, https://www.illumina.com/documents/products/technotes/technote_somatic_variant_ca ller.pdf ). We filtered out variants with variant allele frequencies below 1% and above 35% as potential sequencing noise and germline variation, respectively.
Somatic variant caller
Manufactured by Illumina
The Somatic Variant Caller is a bioinformatics software tool designed for the identification and analysis of somatic genetic variants from next-generation sequencing data. It is capable of detecting single nucleotide variants, insertions, and deletions in tumor samples compared to matched normal samples.
Automatically generated - may contain errors
Lab products found in correlation
Variantstudio software, by Illumina (2 mentions)
Variantstudio 3, by Illumina (1 mentions)
Trusight tumor 15, by Illumina (1 mentions)
Miniseq high output reagent kit, by Illumina (1 mentions)
Variant interpreter software, by Illumina (1 mentions)
Truseq custom amplicon low input kit, by Illumina (1 mentions)
Agilent bioanalyzer system, by Agilent Technologies (1 mentions)
Nextera xt protocol, by Illumina (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Truseq amplicon cancer panel, by Illumina (1 mentions)
Miseq reporter, by Illumina (1 mentions)
Trusight myeloid sequencing panel, by Illumina (1 mentions)
Basespace, by Illumina (1 mentions)
Variantstudio, by Illumina (1 mentions)
Haloplex, by Agilent Technologies (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Real time analysis software, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
11 protocols using somatic variant caller
1
Targeted Amplicon Sequencing for Variant Detection
2
Illumina TruSight Tumor 15 Profiling
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Library preparation was conducted using the Illumina TruSight Tumor 15 covering 15 genes, which are frequently mutated in solid tumors. Subsequent sequencing of pooled libraries was performed in several runs on the MiniSeq Illumina platform using MiniSeq High Output Reagent Kit (300-cycles). Data analysis was conducted using on-instrument Local Run Manager (LRM) Software with TruSight Tumor 15 analysis module. Passed-filter reads were aligned to human reference genome UCSC hg19 using banded Smith Waterman algorithm. Variants were called using Somatic Variant Caller developed by Illumina. All vcf-datasets were annotated using the Illumina VariantStudio 3.0 Software. Across all samples, several hotspot codons were manually evaluated using the Integrative Genomics Viewer (IGV) for potential low-abundance variants (0.1> VAF <2.0%). Annotated plasma variants had to have allele frequencies above a background threshold of the mean of our control samples (three different non-PDAC cfDNA samples).
3
Germline and Somatic Variant Analysis Protocol
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Reads were aligned to the human reference genome (hg19) and variants were called using Somatic Variant Caller (Illumina). Variants identified for each patient were filtered and prioritized using Variant Interpreter Software (Illumina). The analysis of germline alleles (defined as an alternative allele found in ≥35% reads) and somatic variants (defined as an alternative allele seen in 5%–34% reads) was restricted to regions covered at >50× and >200× depth, respectively.
To identify candidate variants, we first selected nonsynonymous and splice site variants. These were then cross‐referenced against the databases ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ ; accessed [19/07/21]) and InFevers (https://infevers.umai‐montpellier.fr/ ; accessed [19/07/21]) to identify known pathogenic or likely pathogenic alleles.16 , 17 , 18 , 19 , 20 In parallel, variants were annotated according to the minor allele frequency reported on the gnomAD for the corresponding ancestry group. Rare variants (minor allele frequency <0.01) were then subjected to in silico pathogenicity prediction using the Combined Annotation Depletion Dependent tool.21 , 22 A scaled Combined Annotation Depletion Dependent score >10 was considered evidence of deleterious potential.22
To identify candidate variants, we first selected nonsynonymous and splice site variants. These were then cross‐referenced against the databases ClinVar (
4
BRCA Gene Sequencing Protocol
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Based on the presented results we aimed to focus on the analysis of both BRCA genes of interest and therefore used the TruSeq Custom Amplicon Low Input Kit (Illumina) in combination with a custom-designed BRCA gene panel. Selected FFPE samples were subjected to dual-pool amplicon-based library preparation. Subsequent sequencing of pooled libraries was performed on the MiniSeq sequencing platform using MiniSeq High Output Reagent Kit for 300-cycles.
Paired-end sequencing resulted in average 6115644.86 (6.1 Mio) paired-end passed filter reads per sample and mean amplicon coverage of 6774. Data analysis was conducted using on board Amplicon DS pipeline. Sequencing data was aligned to the reference genome UCSC hg19 using banded Smith–Waterman algorithm and variant calling was performed with Illumina Somatic variant caller. Filtering of all datasets was conducted manually according to predefined (custom) criteria.
Paired-end sequencing resulted in average 6115644.86 (6.1 Mio) paired-end passed filter reads per sample and mean amplicon coverage of 6774. Data analysis was conducted using on board Amplicon DS pipeline. Sequencing data was aligned to the reference genome UCSC hg19 using banded Smith–Waterman algorithm and variant calling was performed with Illumina Somatic variant caller. Filtering of all datasets was conducted manually according to predefined (custom) criteria.
5
Targeted Deep Sequencing of CD19 Gene
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Exon 1 to 4 including introns of the CD19 locus (chr16:28942047–28944969) was amplified with the Expand long template Polymerase system (Roche). Paired-end libraries were created following the Nextera XT protocol (Illumina) which uses transposome to fragment and immediately tag the DNA with adapter sequences in a single step. Quality of the libraries was proofed using an Agilent Bioanalyzer System (Agilent Technologies). Libraries were subjected to deep sequencing on an Illumina MiSeq sequencer using 151 cycles (paired-end). Minimum depth of coverage was 1000x. Data were processed using BWA Enrichment v1.0 for generation of BAM files and the somatic variant caller of Illumina, which allows to detect low-frequency mutations (below 5%). Analysis of variants was performed with the VariantStudio software (Illumina). Variants with a population frequency less than 5% were analyzed further. Reads were visualized using the IGV software.
6
Targeted Somatic Variant Detection
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The samples were processed on the MiSeq platform with a TruSeq Amplicon Cancer Panel (Illumina, Inc.), which is designed to detect well-known somatic mutations of 48 genes (Table S2). Somatic variants were called using the Somatic Variant Caller (Illumina, Inc.) and annotated using the ANNOVAR (Wang et al., 2010 (link)) software tool (http://www.openbioinformatics.org/annovar/ ) and in-house scripts.
7
Comprehensive Variant Identification Pipeline
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Data analysis was performed using in-house bioinformatic pipelines at the Oxford Molecular Diagnostic Center. In short, FASTQ files were processed by the MiSeq Reporter, and sequences were aligned to the reference genome (hg19) by the Smith-Waterman algorithm and Illumina Somatic Variant caller (1.1.0). Variants were annotated using Illumina VariantStudio (2.2) and were filtered using the following passing filters: quality >100 and consequence: missense, frameshift, stop gained, stop lost, initiator codon, in-frame insertion, in-frame deletion, and splice filters. Variants were first verified by visual check by using the Interactive Genomics Viewer (IGV, 2.3.40) and confirmed by either Sanger sequencing or PCR-based mutational analysis performed in the Great Ormond Street Hospital diagnostic laboratory.
8
Targeted Sequencing of Myeloid Malignancies
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Genomic DNA libraries were prepared using either a custom Haloplex (Agilent) panel, as previously described [21 (link)], or the TruSight Myeloid Sequencing Panel (Illumina) according to the manufacturer’s instructions. Sequencing was carried out on the Illumina NextSeq 500 platform with 150-bp paired-end reads. The resulting FASTQ files were aligned using the TruSeq Amplicon App (version 3.0.0) on BaseSpace (Illumina) and variants called using the Somatic Variant Caller (Illumina). The vcf files generated were filtered in Variant Studio (Illumina) and further verified using the Integrative Genomics Viewer (Broad Institute).
9
MiSeq Sequencing and Variant Calling
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Pooled libraries were sequenced on a MiSeq instrument using a 2 × 150 paired-end format using the Custom Amplicon workflow. Base calls were generated on-instrument with the Real Time Analysis (RTA) software (Illumina). Reads were aligned to the Homo Sapiens – UCSC (h19) genome assembly and the Somatic Variant Caller (Illumina) was used for identification of variants. The Illumina Variant Studio software was used to annotate all detected variants. All alternate variant calls were required to have Q scores of at least Q30 and occur at a frequency of ≥ 15%.
10
Comprehensive Genomic Profiling for Suspected ET
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Demographic characteristics (age and gender) and clinical information (year of diagnosis, cardiovascular risk factors, blood parameters, bone marrow morphology, treatment and clinical outcomes) were determined. Bone marrow biopsy material was only available in 25 of the 35 cases and was reviewed blinded by an independent haematologist. The remaining 10 cases did not have a bone marrow but were considered likely to have a diagnosis of ET because in the past in the real world setting patients with a persistent elevated platelet count and no other cause have had a diagnosis of ET. 4 Suspected TN-ET patients were included after excluding JAK2V617F, CALR exon 9 and MPL exon 10 mutations by conventional techniques (allelic specific PCR, DNA fragment analysis and Sanger sequencing, respectively). 5, 6 Next Generation Sequencing Genomic DNA was extracted from whole peripheral blood and retested for JAK2V617F, CALR exon 9 and MPL exon 10 mutations by conventional techniques. NGS was performed using the Illumina MiSeqTM platform and the Trusight Myeloid gene panel. Sequence variants were called using Somatic Variant Caller (Illumina), with variant filtering using a custom Classification Tree within the Cartagenia BENCH® software. Reporting required total read depth >100X for known variants and >300X for novel variants and allelic frequencies >5%.
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