BIOTECHNOLOGY 69
Validating next-generation sequencing-based genetic tests
Frederick R. Blattner & Tim Durfee showcase software for auto- analysis of internal benchmarking controls.
A
s next-generation sequencing (NGS) makes genetic testing for a wide range of human diseases increasingly commonplace, facile methods for validating the efficacy of those tests are essential.
In the USA, federal regulatory standards embodied in the Clinical Laboratory Improvement Amendments (CLIA), for instance, are designed to ensure that these tests reliably achieve certain performance specifications in terms of accuracy, precision and analytical sensitivity and specificity.
To facilitate the validation process, the National Institute of Standards and Technology (NIST) through the Genome in a Bottle Consortium (GIAB) developed a highly curated set of genome-wide reference materials for the HapMap/1,000 Genomes CEU female, NA12878. Tese materials include BED and VCF files of
Fig. 1. DNASTAR’s dedicated data flow.
high confidence sequence regions and variant calls, respectively. NA12878 genomic DNA and a cell line are available (Coriell Institute), providing laboratories with an internal control for their processing and analysis pipeline. Comparing testing results to the GIAB call sets allows establishment of both the analytical performance for regulatory certification as well as the appropriate assembly thresholds to apply when considering potential variants in patient samples.
Computational challenge For clinical sequencing laboratories to efficiently leverage resources such as the GIAB call sets, assembly and analysis software must support the unique aspects of validation control processing.
For example, analyses should be restricted to the intersection between the GIAB high confidence regions and the target regions of the specific
test. Variant reporting should also match GIAB call set conventions wherever possible to avoid underestimating the accuracy.
Additionally, data processing and statistical calculations should be rapid, automated and reported in an easily interpreted form. Most clinical laboratories lack the bioinformatics expertise needed to build software pipelines capable of handling these challenges.
Software workflow DNASTAR has developed a dedicated workflow within its Lasergene Genomics suite for clinical sequencing labs to utilise the NA12878 reference materials to validate their NGS-based genetic tests (Fig. 1.). Within the SeqMan NGen wizard, users specify: NGS reads from their processed NA12878 sample; the human genome reference version (NA12878 reference materials are in GRCh37 coordinates; an intersected BED file between their targeted regions and
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