70 BIOTECHNOLOGY
GIAB’s NA12878 high confidence regions; and GIAB’s NA12878 VCF file of high confidence variant calls. At runtime, the VCF is filtered down to those positions delineated by the intersected BED file.
Te data is then assembled against the human genome reference sequence using SeqMan NGen running on a standard desktop computer. Fully gapped alignments are analysed in-stream using a modified version of the MAQ variant caller to produce variant and reference call files for each position in the intersected BED file. Key metrics, including the depth of coverage and probability scores, are recorded for each position. Assemblies can be visualised in SeqMan Pro, allowing evidence for variants and regions of low coverage to be assessed.
For accuracy calculations, variant and reference call files are automatically loaded with the
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filtered VCF file into ArrayStar post-assembly. Only positions within the intersected BED are considered. Positions are classified as: true positives (TP), called variants also present in the VCF file; false positives (FP), called variants not in the VCF file; true negatives (TN), called reference bases not in the VCF file; and false negatives (FN), called reference bases that are present in the VCF file. Te counts of each class are then used to calculate various accuracy metrics, including the true positive rate (TPR, “sensitivity”), the true negative rate (TNR, “specificity”) and the false discovery rate (FDR).
Summary report A summary report is produced with the absolute number of positions in each class and the corresponding statistics stratified based on two thresholds: minimum depth of coverage that a position must match or exceed to be considered in the analysis; and
minimum p-value a position must have to be considered a variant. Tree p-values (“Pnotref” of 90.0%, 99.0% and 99.9% corresponding to phred-like scores of 10, 20 and 30, respectively) are employed for each of 12 different depths of coverage cutoffs (ranging from 1 to 100). Te number and percentage of targeted bases meeting each depth cutoff are also presented.
Lessons learned NGS-based genetic tests promise to greatly enhance patient care in this era of personalised medicine. Given their potential impact on treatment decisions, it is critical that those tests be appropriately validated as part of their regulatory approval and as a measure of routine assessment of the test’s performance. DNASTAR’s validation control workflow using NIST/GIAB’s NA12878 reference materials greatly facilitates this process.
For more information ✔ at
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Screenshot from Lasergene Genomics Suite
Frederick R. Blattner & Tim Durfee are with DNASTAR.
www.dnastar.com
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