Reviewing the quality of reads by IGV
Raw sequencing data were reviewed for the detected variants using
Integrative Genomic Viewer (IGV) software
(https://software.broadinstitute.org/software/igv/)
according to its instructions. Briefly, BAM files were imported to IGV
and the quality of sequencing reads, coverage and number of the reads
(depth) for the given variants were reviewed.
Variant Confirmation and Segregation
Analysis
The reported positive findings in group A were reviewed with regard to
the test setting (research or clinical), applied technique (NGS-based
methods or Sanger sequencing), and the variant characteristics, to
indicate whether further confirmatory tests were required. As a result,
patients that were studied in a research setting, or had INDEL variants
identified by WES, were investigated by Sanger verification. Although
group B patients were studied by targeted-capture panel with high
coverage depth, a subset of variants with depressed allele fraction
(<30% of alternate allele), particularly deletion/insertion
type variants, underwent additional validation with Sanger sequencing
which was performed on an ABI3130 (Life Technologies) according to the
standard procedures. Amplification primers were either selected from
published papers (if available) or designed using Primer 3 software.
Segregation analysis was carried out when additional family members were
available and segregation data was helpful for classification and
interpretation of the variants, especially variants with unknown
clinical significance.
Complementary Sanger
Sequencing
In case of no positive finding in target NGS, raw sequencing data were
reviewed in each patient using IGV software, and low coverage regions
were then sequenced by Sanger sequencing.