Genetic analysis was carried out on genomic DNA from blood by clinical exome sequencing (Trusight One panel, Illumina, San Diego, CA, USA), using a NextSeq 500 platform, with 20× average sequencing coverage. Read files were obtained from the Illumina platform through the manufacturer’s proprietary software (Illumina VariantStudio Software v3.0, Illumina, San Diego, CA, USA). Variant validation and segregation analysis were performed by Sanger sequencing. In silico analysis of the functional impact of the identified LAMB3 mutations was performed by NNSPLICE (0.9 version) (https://www.fruitfly.org/seq_tools/splice.html , accessed on 24 January 2019) and Combined Annotation Dependent Depletion (CADD 1.3) (http://pec630.rockefeller.edu:8080/MSC/ , accessed on 23 April 2021) prediction software program. Total RNA from patient keratinocytes was reverse transcribed (Superscript III RT, Thermo Fisher Scientific, Carlsbad, CA, USA) and amplified with oligonucleotides 5’-GCTGAGGCTGAGGAAGCCAG-3’ (forward primer) and 5’-TGTAACTGTCCCATTGGCTC-3’ (reverse primer) to generate a fragment encompassing the two parental variants [20 (link)]. The resulting PCR product was then cloned in a pCR2.1 vector (TA cloning kit, Thermo Fisher Scientific), and individual subclones were sequenced.
Variantstudio software v3
Manufactured by Illumina
Sourced in United States
VariantStudio Software v3.0 is a tool for analyzing and interpreting DNA sequence variation data. It provides a user-friendly interface for visualizing, annotating, and filtering genetic variants from next-generation sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Miseq sequencer, by Illumina (2 mentions)
Ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
Trusight one panel, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Ampliseq library plus, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Qiaamp dna ffpe generead kit, by Qiagen (1 mentions)
Qubit 1 dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Miseq device, by Illumina (1 mentions)
Basespace variant interpreter, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Multiplex kit, by Qiagen (1 mentions)
Easytaq dna polymerase, by Transgene (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Thermo Fisher Scientific (1 mentions)
16s metagenomic sequencing library preparation, by Illumina (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
7 protocols using variantstudio software v3
1
Genetic Analysis of LAMB3 Mutations
2
Comprehensive Genetic Analysis Protocol
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The NGS generated for each cluster a forward and a reverse read. Read1 and Read2 are defined as read pairs. The sequencing run was valid when both Read1 and Read2 had at least 220 bp. The bioinformatic evaluation comprised of three parts -alignment of obtained reads to .bam files (Assembly GRCh37), secondary data analysis getting the variant annotation in the .vcf files and tertiary data analysis resulting in interpretation of variants. The sequence alignment was performed with MiSeq Reporter Software v2.5.1.3 (Illumina, USA) and the .vcf files were generated with Illumina Variant Studio Software v 3.0 (Illumina, USA). Sequences were annotated starting with the first nucleotide that corresponds for the first A in the first translated amino acid in coding reference cDNA. The cDNA reference sequences for MHL1, MSH2, MSH6 and PMS2 were NM_000249.3, NM_000251.2, NM_000179.2 and NM_000535.5, respectively. The potential pathogenic variant was compared with free accessible database Clin-Var, Human Gene Mutation Database (http://www.hgmd .cf.ac.uk/ac/all.php), Ensembl (https://www.ensembl.org /Homo_sapiens/Transcript/ProtVariations/), the InSight databases (https://www.insight-group.org/variants/data-bases/) and published literature. The presence of pathogenic variant was confirmed by Sanger sequencing.
3
Exonic Sequencing of IDH1 and IDH2 in FFPE Tumor Samples
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DNA from 4–8 sections (4–5 μm each) of formalin-fixed paraffin-embedded (FFPE) tumor samples (which were selected by a pathologist as described above) was extracted using the “QIAamp DNA FFPE GeneRead Kit” (Catalogue Reference: 180134; Qiagen, Germantown, MD, USA). Subsequently, the whole exonic region of IDH1 and IDH2 genes was then sequenced using an Illumina MiSeq device (version 3.1.0.13) following the manufacturer’s instructions, as previously described [39 (link)]. In brief, after DNA quantification from FFPE tumor samples using Qubit 1× dsDNA HS Assay Kit (Catalogue Reference: Q33230; Thermo Fisher Scientific, Waltham, MA, USA), 50–150 ng was used for mutational analysis by AmpliSeq methodology (Illumina, Inc., San Diego, CA, USA). AmpliSeq Library PLUS was used for library preparation (Catalogue Reference: 20019101; Illumina, Inc., USA), followed by the amplification of target regions and second amplification of libraries, which were diluted and denatured for bridge clonal amplification and paired-end sequencing using MiSeq Reagent kit v2 (300-cycles) (Catalogue Reference: MS-102-2002; Illumina, Inc., USA) in a MiSeq instrument (Illumina, Inc., USA). Variant calling files annotation, and the identification and classification of detected genetic variants were performed with the VariantStudio software v3.0 (Illumina, Inc., USA).
4
Variant Prioritization and Annotation for HCM
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We used the Illumina VariantStudio Software v3.0 (Illumina, San Diego) and BaseSpace Variant Interpreter (Illumina, San Diego) for variant prioritization and annotation. Variants with read depth < 10×, quality < 200, synonymous and intronic variants in non-splice regions and minor allele frequency (MAF) higher than expected for HCM [15 (link)] were removed for further analysis. We used Sorting Tolerant From Intolerant (SIFT) and PolyPhen-2 prediction tools to assess the potential functional impacts of variants and detect missense variants. We used the National Center for Biotechnology Information (NCBI) ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/ ) and Human Gene Mutation Database (HGMD) databases to assess the clinical impact of variants [16 (link)]. Potential variants were classified according to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines into one of the following classes: pathogenic, likely pathogenic, uncertain significance, likely benign and benign with respect to HCM [17 (link)]. All pathogenic and likely pathogenic variants were validated using DNA Sanger sequencing.
5
HLA-G Gene Sequencing and Haplotyping
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Full-length NGS of the HLA-G gene including the 5’URR, introns, and 3’UTR, was performed using the MiSeq platform (Illumina). The HLA-G gene region extending from positions −1550 to 3404, relative to the start codon, was analyzed using long-range PCR. Specific primers were designed in relation to HLA-G RefSeqGene version NG_029039.1 (NCBI database), as previously described (58 (link)). The libraries were prepared starting from 1 ng of PCR product using the Nextera XT DNA Library Preparation Kit. The normalized libraries (4 nM) were pooled and loaded onto V3 flow cells for 600 cycles of bidirectional sequencing in a MiSeq Illumina Sequencer. The automatically generated FASTQ files were then processed with the MiSeq Reporter v2.6 for alignment and variant calling, and VariantStudio Software v3.0 for variant classification (Illumina, Netherlands). The variants were verified individually and then inserted into appropriate electronic spreadsheets for statistical analysis (see below). HLA-G 3’UTR haplotypes were determined by nucleotide sequence variations from +2945 to +3259 nucleotides, using a methodology and a nomenclature described elsewhere (14 (link), 59 (link)).
6
16S Metagenomic Sequencing and TERT Profiling
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The PCR amplicon was performed following the protocol ‘16S Metagenomic Sequencing Library Preparation’ (Illumina). First, the regions with hotspot mutations in the TERT promoter was amplified by PCR. The sequences of the primers used are listed in Supporting Information Methods Table S2 . Amplicon PCR was performed using the DNA polymerase enzyme from the Multiplex kit (QIAGEN) following the manufacturer's instructions. PCR products were purified using AMPure XP beads (Beckman Coulter). Index PCR was later performed with the EasyTaq DNA polymerase (TransGen Biotech) using synthetic indexes from the Nextera XT Index kit (Illumina). Index PCR products were purified with AMPure XP beads and quantified by PicoGreen (Thermo Fisher Scientific). The concentration of each amplicon was normalized, and library pooling was performed by mixing equal volumes of each amplicon with unique indexes.
Libraries were sequenced on an MiSeq sequencer (Illumina), following the protocol provided by the manufacturer. The sequencing module used was the ‘PCR Amplicon’ protocol with a paired‐end design with 150 base pairs reads of length. The reference genome used for reads alignment was GRCh37 and Illumina Variant Studio software v3.0 was used for the annotation of the mutations.
Libraries were sequenced on an MiSeq sequencer (Illumina), following the protocol provided by the manufacturer. The sequencing module used was the ‘PCR Amplicon’ protocol with a paired‐end design with 150 base pairs reads of length. The reference genome used for reads alignment was GRCh37 and Illumina Variant Studio software v3.0 was used for the annotation of the mutations.
7
Variant Analysis Using VariantStudio and IGV
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Data were analyzed by VariantStudio software v.3.0 (Illumina, San Diego, CA, USA) and the Integrative Genomics Viewer (IGV) was used as a visualization tool for the detected variants. We used theavailable online dbSNP database, (https://www.ncbi.nlm.nih.gov/snp/ ) and Ensembl browser (http://grch37.ensembl.org/index.html ) (accessed on 3 June 2021) to find the location and frequency of the variants.
Variant classification was performed following criteria of the American College of Medical Genetics and Genomics (ACMG) guidelines.
Variant classification was performed following criteria of the American College of Medical Genetics and Genomics (ACMG) guidelines.
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