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Technical in-depth comparison of two massive parallel DNA-sequencing methods for formalin-fixed paraffin-embedded tissue from victims of sudden cardiac death
Örebro universitet, Institutionen för medicinska vetenskaper. Department of Laboratory Medicine, Örebro University Hospital, Sweden.ORCID-id: 0000-0002-7954-0696
Örebro universitet, Institutionen för medicinska vetenskaper. Department of Laboratory Medicine, Örebro University Hospital, Sweden.
Division of Drug Research, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden; Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
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2021 (Engelska)Ingår i: Forensic Science International: Genetics, ISSN 1872-4973, E-ISSN 1878-0326, Vol. 53, artikel-id 102522Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively. In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at ≥ 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6-100%), and high precision, 99.9% (range 99.5-100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2-91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8-98.7%) and high precision, 99.9% (range 99.3-100%) in all analyzed samples, including the severely fragmented samples.

Ort, förlag, år, upplaga, sidor
Elsevier, 2021. Vol. 53, artikel-id 102522
Nyckelord [en]
DNA mutational analysis/methods, Death, Sudden, Cardiac, Massive parallel sequencing, MPS, Paraffin embedding, Sequence analysis, DNA, Tissue fixation
Nationell ämneskategori
Kardiologi
Identifikatorer
URN: urn:nbn:se:oru:diva-91666DOI: 10.1016/j.fsigen.2021.102522ISI: 000670126400008PubMedID: 33945952Scopus ID: 2-s2.0-85104938164OAI: oai:DiVA.org:oru-91666DiVA, id: diva2:1553270
Anmärkning

Funding Agencies:

ALF funding Region Örebro County  

Örebro County Council Research committee  

Tillgänglig från: 2021-05-07 Skapad: 2021-05-07 Senast uppdaterad: 2021-08-02Bibliografiskt granskad

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Adolfsson, EmmaQvick, AlvidaGreen, Anna
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Institutionen för medicinska vetenskaperRegion Örebro län
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Forensic Science International: Genetics
Kardiologi

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