Genomic DNA Isolation and Variant Analysis

Plasma blood samples were collected in Vacutainer tubes. Subsequently, genomic DNA was isolated from whole blood using the QIAsymphony DSP DNA Mini Kit (Qiagen, Germantown, MD, USA) and used to prepare indexed libraries using the Trusight Cancer Panel – Nextera DNA Flex Pre-Enrichment Library Prep (Illumina, San Diego, USA). Libraries were sequenced on a MiSeq genetic analyzer (Illumina, Inc., San Diego, CA), according to the manufacturer’s protocols. Annotation was performed against the human reference genome GRCh38 using VariantStudio V.3 (Illumina, Inc., San Diego, CA). Based on the data generated from this software, alterations were identified as pathogenic when classified as disease causing or as variants of unknown significance (VUS) when evidence regarding their pathogenicity was either conflicting or limited. The validation of results was performed according to criteria of American College of Medical Genetics – ACMG (8 (link)) and National Comprehensive Cancer Network (NCCN) guidelines.

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Andrikopoulou A., Zografos E., Apostolidou K., Kyriazoglou A., Papatheodoridi A.M., Kaparelou M., Koutsoukos K., Liontos M., Dimopoulos M.A, & Zagouri F. (2022). Germline and somatic variants in ovarian carcinoma: A next-generation sequencing (NGS) analysis. Frontiers in Oncology, 12, 1030786.

Publication 2022

QIAsymphony DSP DNA Mini Kit MiSeq genetic analyzer VariantStudio V.3

Blood Cancer Dna library Genome human Genomic Miseq Pathogenicity Plasma

Corresponding Organization : National and Kapodistrian University of Athens

Top 5 similar protocols

Variable analysis

independent variables

Plasma blood sample collection in Vacutainer tubes
Genomic DNA isolation from whole blood using the QIAsymphony DSP DNA Mini Kit (Qiagen, Germantown, MD, USA)
Preparation of indexed libraries using the Trusight Cancer Panel – Nextera DNA Flex Pre-Enrichment Library Prep (Illumina, San Diego, USA)
Sequencing of libraries on a MiSeq genetic analyzer (Illumina, Inc., San Diego, CA)

dependent variables

Identification of alterations classified as pathogenic or variants of unknown significance (VUS)

control variables

Annotation against the human reference genome GRCh38 using VariantStudio V.3 (Illumina, Inc., San Diego, CA)
Validation of results according to criteria of American College of Medical Genetics – ACMG and National Comprehensive Cancer Network (NCCN) guidelines

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

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