Targeted next-generation sequencing (NGS) of nine candidate genes with custom AmpliSeq in patients and a cardiomyopathy risk group

Glotov, Andrey S.; Казаков, С. В.; Zhukova, Elena; Alexandrov, A. A.; Glotov, Oleg S.; Pakin, Vladimir S.; Danilova, Maria M.; Poliakova, Irina V.; Niyazova, S. S.; Dolmatovich, T. V.; Комиссарова, С. М.; Курникова, Елена Анатольевна; Sarana, Andrey М.; Sсherbak, Sergey G.; Sergushichev, Alexey; Shalyto, Anatoly; Baranov, V. S.

doi:10.1016/j.cca.2015.04.014

Cited by 29 publications

(20 citation statements)

References 51 publications

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“…Coverage statistics (Table 1) were comparable among all runs and were in the range of accepted quality criteria [40], [41], [42]. During the seven runs, a median of 2.54 × 10 6 reads per run was generated.…”

Section: Resultsmentioning

confidence: 83%

Next-generation sequencing of human opioid receptor genes based on a custom AmpliSeq™ library and ion torrent personal genome machine

Kringel

Lötsch

2016

Clinica Chimica Acta

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BackgroundThe opioid system is involved in the control of pain, reward, addictive behaviors and vegetative effects. Opioids exert their pharmacological actions through the agonistic binding at opioid receptors and variation in the coding genes has been found to modulate opioid receptor expression or signaling. However, a limited selection of functional opioid receptor variants is perceived as insufficient in providing a genetic diagnosis of clinical phenotypes and therefore, unrestricted access to opioid receptor genetics is required.MethodsNext-generation sequencing (NGS) workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes related to the opioid receptor group (OPRM1, OPRD1, OPRK1, SIGMA1, OPRL1) on an Ion PGM™ Sequencer. A cohort of 79 previously studied chronic pain patients was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding. In-silico analysis was performed using SNP and Variation Suite® software.ResultsThe amplicons covered approximately 90% of the target sequence. A median of 2.54 × 106 reads per run was obtained generating a total of 35,447 nucleotide reads from each DNA sample. This identified approximately 100 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19, including functional variants such as the OPRM1 rs1799971 SNP (118 A > G) as the most scientifically regarded variant or rs563649 SNP coding for μ-opioid receptor splice variants. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.ConclusionResults suggested that the NGS approach based on AmpliSeq™ libraries and Ion PGM sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of opioid receptor genes. The method includes the variants studied so far for functional associations and adds a large amount of genetic information as a basis for complete analysis of human opioid receptor genetics and its functional consequences.

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Section: Resultsmentioning

confidence: 83%

Next-generation sequencing of human opioid receptor genes based on a custom AmpliSeq™ library and ion torrent personal genome machine

Kringel

Lötsch

2016

Clinica Chimica Acta

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“…The sequencing of the whole cohort required two separate runs with each 40 patients’ samples. Coverage statistics (Table 1) were comparable between both runs and were in the range of accepted quality criteria [20–22]. During the runs, a median of 2.81 · 10 6 reads per run was generated.…”

Section: Resultsmentioning

confidence: 92%

Next-generation sequencing of the human TRPV1 gene and the regulating co-players LTB4R and LTB4R2 based on a custom AmpliSeq™ panel

et al. 2017

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BackgroundTransient receptor potential cation channel subfamily V member 1 (TRPV1) are sensitive to heat, capsaicin, pungent chemicals and other noxious stimuli. They play important roles in the pain pathway where in concert with proinflammatory factors such as leukotrienes they mediate sensitization and hyperalgesia. TRPV1 is the target of several novel analgesics drugs under development and therefore, TRPV1 genetic variants might represent promising candidates for pharmacogenetic modulators of drug effects.MethodsA next-generation sequencing (NGS) panel was created for the human TRPV1 gene and in addition, for the leukotriene receptors BLT1 and BLT2 recently described to modulate TRPV1 mediated sensitisation processes rendering the coding genes LTB4R and LTB4R2 important co-players in pharmacogenetic approaches involving TRPV1. The NGS workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes on an Ion PGM™ Sequencer. A cohort of 80 healthy subjects of Western European descent was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding.ResultsThe amplicons covered approximately 97% of the target sequence. A median of 2.81 x 106 reads per run was obtained. This identified approximately 140 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19 comprising the three genes TRPV1, LTB4R and LTB4R2. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.ConclusionsResults suggested that the NGS approach based on AmpliSeq™ libraries and Ion Personal Genome Machine (PGM) sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of TRPV1 and functionally related genes. The method adds a large amount of genetic information as a basis for complete analysis of TRPV1 ion channel genetics and its functional consequences.

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“…At a molecular level, we identified a pathogenic heterozygous variant in FGFR2 NM_000141. 4 Continued www.nature.com/scientificreports www.nature.com/scientificreports/ ClinVar: 374823) (see Fig. 3d,e).…”

Section: Identification and Evaluation Of Candidate Variantsmentioning

confidence: 99%

Adapting SureSelect enrichment protocol to the Ion Torrent S5 platform in molecular diagnostics of craniosynostosis

Bukowska‐Olech

Popiel²,

Koczyk

et al. 2020

Sci Rep

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Obtaining reliable and high fidelity next-generation sequencing (NGS) data requires to choose a suitable sequencing platform and a library preparation approach, which both have their inherent assayspecific limitations. Here, we present the results of successful adaptation of SureSelect hybridisationbased target enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is designed to work preferably with amplicon-based panels. In our study, we applied a custom NGS panel to screen a cohort of 16 unrelated patients affected by premature fusion of the cranial sutures, i.e. craniosynostosis (CS). CS occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. The approach presented here allowed us to achieve high quality NGS data and confirmed molecular diagnosis in 19% of cases, reaching the diagnostic yield similar to some of the published research reports. In conclusion, we demonstrated that an alternative enrichment strategy for library preparations can be successfully applied prior to sequencing on the Ion Torrent S5 platform. Also, we proved that the custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS. Next-Generation Sequencing (NGS) in Medical Genetics Routine NGS diagnostics requires high-quality sequencing data, short turnaround time and reasonable cost of the investigations. Therefore, out of the three major NGS-based diagnostic strategies, i.e. whole exome sequencing (WES), whole genome sequencing (WGS), and targeted gene panel sequencing, the final approach is ubiquitous and broadly applied in the clinical settings 1-5. Successful implementation of targeted NGS in medical diagnostics results from several advantages. First, it generates disease-restricted data with fewer variants of uncertain significance, simplifying the analysis. Next, it provides very high coverage and read depth of selected regions, and finally, it limits the need for expensive laboratory equipment and data storage 6,7. In order to generate reliable, high fidelity NGS data one has to choose a suitable sequencing platform and a library preparation protocol 6,8. Different NGS platforms are known to have their specific limitations, such as underrepresentation of sequences with high guanine-cytosine (GC) content in case of Illumina or homopolymer length estimation bias in Ion Torrent semiconductor-based sequencing systems 9-14. In addition to dissimilarities of NGS platforms and their specific inbuilt artefacts, also the sample preparation protocols differ in many aspects, including enrichment strategy. Currently, two major targeted enrichment strategies are available, i.e. PCR-based methods and hybridisation-based protocols. Although targeted PCR-based amplicon approach offers both easy workflow and shorter reaction time, requiring low DNA input at the same time, it suffers from several limitations, such as lower

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Targeted next-generation sequencing (NGS) of nine candidate genes with custom AmpliSeq in patients and a cardiomyopathy risk group

Cited by 29 publications

References 51 publications

Next-generation sequencing of human opioid receptor genes based on a custom AmpliSeq™ library and ion torrent personal genome machine

Next-generation sequencing of human opioid receptor genes based on a custom AmpliSeq™ library and ion torrent personal genome machine

Next-generation sequencing of the human TRPV1 gene and the regulating co-players LTB4R and LTB4R2 based on a custom AmpliSeq™ panel

Adapting SureSelect enrichment protocol to the Ion Torrent S5 platform in molecular diagnostics of craniosynostosis

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