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Abstract

Background

Sequencing technologies have grown exponentially in recent years resulting in next-generation sequencing (NGS) platforms which are more efficient in terms of biochemistry, time and cost. NGS is particularly applicable to highly heterogeneous diseases such as non-syndromic hearing loss (NSHL). Currently, more than 80 genes are clinically relevant and are known to cause hearing loss in humans (Vona B et al., 2015); however, there are about 129 disease associated loci (Vona B et al., 2014).

Aims

The aim of this study is to introduce a molecular diagnostic test in Hamad Medical Corporation, Qatar, using a targeted custom made 80 gene panel on Ion Personal Genome Machine® (PGM™, Thermo Fisher Scientific, USA), in order to obtain rapid and accurate NGS results in compliance with guidelines of the College of American Pathologists (CAP) and the European Molecular Genetics Quality Network.

Methods

Whole blood samples from 91 Qatari individuals (representing 31 unrelated families) that were received by the Molecular Genetics Laboratory for molecular diagnostic purposes since February 2009 were used in this study. DNA was extracted using the Promega Maxwell® 16 Blood DNA purification kit. Libraries were prepared using the Ion AmpliSeq™ Library kit with 200bp chemistry, amplified using emulsion PCR on the Ion OneTouch™ 2 system and enriched using the Ion One Touch™ ES system. Four barcoded libraries (i.e. 4 individuals) were sequenced in parallel on the Ion™ 318 v2 chip using the Ion PGM™. Base calling, signal processing and variant calling were performed using the Torrent Suite™ software. All variants were checked with the Ion Reporter™software and Intergrative Genomics Viewer (IGV, Broad institute) and in silico analysis was done using databases such as ClinVar, dbSNP, 1000 genomes browser, Deafness Variation Database, PolyPhen, SIFT, Human Splicing Finder and MutationTaster. All variants are being confirmed using Sanger sequencing which remains as the gold standard.

Results

We have sequenced 91 affected and unaffected Qatari individuals from 31 unrelated families using the 80 gene panel on the Ion PGM™. On average, 4.1 million on target reads were generated per run with a mean depth of 270 and with mean coverage of 95% at 20X and 90% at 100X. On average about 325 variants were detected per individual. Causative variant(s) were identified based on factors such as pathogenicity or clinical relevance of the variant, minor allele frequency, coverage, quality and variant segregation within the family. The study has identified key players within the Qatari population such as TECTA, CDH23, OTOF, TMC1, TRIOBP and WFS1, with about 65% of the genes resulting in an autosomal recessive mode of inheritance and 35% being autosomal dominant. All missense mutations, including premature stop codon mutations, were covered at >100x whereas indels ranging from 2 to 78 bp were covered at >50x, in line with CAP guidelines. Furthermore, reproducibility of the results was checked by repeat sequencing of individuals at random resulting in up to 97% similarity between the runs. So far we have been able to detect causative mutation(s) in approx. 85% of the cases using the custom panel. For the remaining 15% of the families targeted resequencing and whole exome sequencing were performed in order to determine a genetic cause.

Conclusion

Identification of disease causing variants for diagnosis of heterogenic disorders such as NSHL is challenging and laborious with traditional sequencing technologies. The advent of targeted NGS in molecular diagnostics has reformed the field, providing clinically significant information in a time and cost efficient manner. Our 80-gene custom panel for NSHL on the Ion PGM™ has proved to be a powerful diagnostic tool offering accurate results which is essential for genetic counselling of patients and their family members.

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/content/papers/10.5339/qfarc.2016.HBPP2915
2016-03-21
2019-11-22
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