Customer Publications May 2022

         May 31, 2022

As we look back on May, I wanted to highlight a range of applications that our VarSeq suite is capable of and show the success of our partners. In these publications, our VarSeq suite is utilized for the analysis of whole-exome, clinical variant classification and association, and assisting in an NGS panel for clinical oncology use. VarSeq’s range and capability of annotating and filtering variants can be applied in a broad spectrum with customizability which can be seen in the following publications.

Clinical whole exome sequencing revealed de novo Heterozygous Stop-Gain and Missense variants in the STXBP1 gene associated with Epilepsy in Saudi Families

Intellectual disability and developmental encephalopathies are mostly linked with infant epilepsy. Epileptic encephalopathy is a term that is used to define association between developmental delay and epilepsy. Mutations in the STXBP1 (Syntaxin-binding protein 1) gene have been previously reported in association with multiple severe early epileptic encephalopathies along with many neurodevelopmental disorders. Among the disorders produced due to any mutations in the STXBP1 gene is developmental and epileptic encephalopathy 4 (OMIM: 612164), is an autosomal dominant neurologic disorder categorized by the onset of tonic seizures in early infancy (usually in the first months of life). In this article, we report two Saudi families one with de novo heterozygous stop-gain mutation c.364C > T and a novel missense c. 305C > A p.Ala102Glu in exon 5 of the STXBP1 gene (OMIM: 602926) lead to development of epileptic encephalopathy 4. The variants identified in the current study broadened the genetic spectrum of STXBP1 gene related with diseases, which will help to add in the literature and benefit to the studies addressing this disease in the future.

Muhammad Imran Naseer, Angham Abdulrhman Abdulkareem, Mahmood Rasool, Bader Shirah, Hussein Algahtani, Osama Y. Muthaffar, Peter Natesan Pushparaj, Clinical whole exome sequencing revealed de novo Heterozygous Stop-Gain and Missense variants in the STXBP1 gene associated with Epilepsy in Saudi Families, Saudi Journal of Biological Sciences, 2022, 103309, ISSN 1319-562X, https://doi.org/10.1016/j.sjbs.2022.103309

Development and evaluation of ActSeq: A targeted next-generation sequencing panel for clinical oncology use

The demand for high-throughput genetic profiling of somatic mutations in cancer tissues is growing. We sought to establish a targeted next generation sequencing (NGS) panel test for clinical oncology practice.

Customized probes were designed to capture exonic regions of 141 genes selected for the panel, which was aimed for the detection of clinically actionable genetic variations in cancer, including KRASNRASBRAFALKROS1KIT and EGFR. The size of entire targeted regions is 0.8 Mb. Library preparation used NEBNext Ultra II FS kit coupled with target enrichment. Paired-end sequencing was run on Illumina NextSeq 500 at a read length of 150 nt. A bioinformatics workflow focusing on single nucleotide variant and short insertions and deletions (SNV/indel) discovery was established using open source, in-house and commercial software tools. Standard reference DNA samples were used in testing the sensitivity and precision and limit of detection in variant calling.

The general performance of the panel was observed in pilot runs. Average total reads per sample ranged from 30 million to 48 million, 73% ~82% unique reads. All runs had more than 99% average mapping rate. Mean target coverage ranged from 727x to 879x. Depth of coverage at 50x or more reached 87% of targeted region and 60% of targeted region received 500x or more coverage depth. Using OncoSpan HD827 DNA, which bears 144 variants (SNV/indel) from 80 genes that are within the targeted region on the panel, our somatic variant calling pipeline reached 97% sensitivity and 100% precision respectively, with near 48 million reads. High concordance with orthogonal approaches in variant detection was further verified with 7 cancer cell lines and 45 clinical specimens.

We developed a NGS panel with a focus on clinically actionable gene mutations and validated the performance in library construction, sequencing and variant calling. High concordance with reference materials and orthogonal mutation detection was observed.

Shi Z, Lopez J, Kalliney W, Sutton B, Simpson J, Maggert K, et al. (2022) Development and evaluation of ActSeq: A targeted next-generation sequencing panel for clinical oncology use. PLoS ONE 17(4): e0266914. https://doi.org/10.1371/journal.pone.0266914

POS0164 GENETIC ANALYSIS OF WHOLE EXOME SEQUENCING IN A COHORT OF CHILDREN WITH REFRACTORY JIA REVEALS GENETIC RISK FACTORS FOR RARE JUVENILE DISEASES.

Juvenile idiopathic arthritis (JIA) encompasses a group of heterogeneous rheumatic diseases of childhood onset. JIA can result in long term disability and remission is the main goal of treatment. However refractory disease can occur, which is defined as the absence of response to a standard disease therapy. A genetic basis for refractory disease has yet to be explored, where deleterious rare variants can complicate diagnosis or treatment outcome.

To investigate, through genetic analysis, whether children with JIA that is refractory carry rare genetic risk factors in genes linked to monogenic diseases.

Whole exome sequencing of 99 children with JIA was performed with the Agilent SureSelect Human All ExonV6 kit. All quality control, variant filtering and annotation was performed in Varseq (version 2.2.1). Variants with a read depth <30 and genotype quality <80 were removed. Rarity and pathogenicity filters were then applied to remove variants with an allele frequency >1% (based on ExAC, gnomAD, gnomAD exome, NHLBI and 1KGp phase 3), classified as benign or likely benign on ClinVar, with a CADD PHRED score <15 and a REVEL score >0.7. Variants were annotated if they appeared in a gene from the primary immunodeficiency PanelApp (Martin et al., 2019), in a gene associated with an arthritis phenotype or in a gene that appeared on a paediatric monogenic gene list. The variants were then classified using ACMG guidelines (Richards et al., 2015) and benign, or likely benign, classified variants were removed.

Tordoff M, Smith S, Rice GI, et alPOS0164 GENETIC ANALYSIS OF WHOLE EXOME SEQUENCING IN A COHORT OF CHILDREN WITH REFRACTORY JIA REVEALS GENETIC RISK FACTORS FOR RARE JUVENILE DISEASES.Annals of the Rheumatic Diseases 2022;81:311. http://dx.doi.org/10.1136/annrheumdis-2022-eular.1075

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