Germline Mutation Detection using Ion S5

All the DNA samples were screened for germline mutations using the On-Demand Research Assay on the Ion S5 system (both ThermoFisher Scientific, Waltham, MA, USA). Each DNA sample was checked for concentration using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The concentration of input DNA was then adjusted to 50 pmol.
The library and template preparations were performed using the automated Ion Chef System, then sequenced in Ion S5 with Ion 520 Chip (all Thermo Fisher Scientific) according to the manufacturer’s instructions.
Sequencing results were aligned to the hg19 human reference genome and analyzed using the Ion Reporter Software Version 5.10 (Thermo Fisher Scientific).
The variant frequency cut-off for the detection of gene germline mutation was defined as 20%, as recommended by a previous study [7 (link)].
The On-Demand Research Assay showed 99.85% sensitivity, 100% specificity, 0% false-positive rate and 99.99% accuracy in detecting single nucleotide variations (SNVs) and small deletions.

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Velázquez C., Lastra E., Avila Cobos F., Abella L., de la Cruz V., Hernando B.A., Hernández L., Martínez N., Infante M, & Durán M. (2020). A comprehensive custom panel evaluation for routine hereditary cancer testing: improving the yield of germline mutation detection. Journal of Translational Medicine, 18, 232.

Publication 2020

Ion S5 system Qubit 3.0 Fluorometer Ion S5 Ion 520 Chip Ion Reporter Software Version 5.10

Assay Chip Deletions Genome human Germline mutation Library Nucleotide Sensitivity

Corresponding Organization : Instituto de Biomedicina y Genética Molecular de Valladolid

Other organizations : Hospital Universitario de Burgos, Ghent University Hospital, Hospital Universitario Río Hortega

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Variable analysis

independent variables

Screening of DNA samples for germline mutations using the On-Demand Research Assay on the Ion S5 system

dependent variables

Detection of single nucleotide variations (SNVs) and small deletions

control variables

Concentration of input DNA adjusted to 50 pmol
Library and template preparations performed using the automated Ion Chef System
Sequencing performed on Ion S5 with Ion 520 Chip
Sequencing results aligned to the hg19 human reference genome
Variant frequency cut-off for germline mutation detection set at 20%

controls

The On-Demand Research Assay showed 99.85% sensitivity, 100% specificity, 0% false-positive rate and 99.99% accuracy in detecting single nucleotide variations (SNVs) and small deletions, which can be considered as positive controls.

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