Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing

Muscular Dystrophies (MDs) are a group of inherited diseases and heterogeneous in nature. To date, 40 different genes have been reported for the occurrence and/or progression of MDs. This study was conducted to demonstrate the application of next-generation sequencing (NGS) in developing a time-saving and cost-effective diagnostic method to detect single nucleotide variants (SNVs) and copy number variants (CNVs) in a single test. A total of 123 cases clinically suspected of MD were enrolled in this study. Amplicon panel-based diagnosis was carried out for 102 (DMD/BMD) cases and the results were further screened using multiplex ligation-dependent probe amplification (MLPA). Whilst in the case of LGMD (N = 19) and UMD (N = 2), only NGS panel-based analysis was carried out. We identified the large deletions in 74.50% (76/102) of the cases screened with query DMD or BMD. Further, the large deletion in CAPN3 gene (N = 3) and known SNV mutations (N = 4) were identified in LGMD patients. Together, the total diagnosis rate for this amplicon panel was 70.73% (87/123) which demonstrated the utility of panel-based diagnosis for high throughput, affordable, and time-saving diagnostic strategy. Collectively, present study demonstrates that the panel based NGS sequencing could be superior over to MLPA.

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Linked-Read sequencing resolves complex structural variants

10.1101/231662 ◽

2017 ◽

Cited By ~ 4

Author(s):

Sarah Garcia ◽

Stephen Williams ◽

Andrew Wei Xu ◽

Jill Herschleb ◽

Patrick Marks ◽

...

Keyword(s):

Chromosomal Rearrangements ◽

Copy Number Variants ◽

Cost Effective ◽

Sequencing Depth ◽

Structural Variants ◽

Single Nucleotide Variants ◽

Candidate Sequence ◽

Full Spectrum ◽

Short Reads ◽

Complex Structural

SummaryLarge genomic structural variants (>50bp) are important contributors to disease, yet they remain one of the most difficult types of variation to accurately ascertain, in part because they tend to cluster in duplicated and repetitive regions, but also because the various signals for these events can be challenging to detect with short reads. Clinically, aCGH and karyotype remain the most commonly used assays for genome-wide structural variant (SV) detection, though there is clear potential benefit to an NGS-based assay that accurately detects both SVs and single nucleotide variants. Linked-Read sequencing is a relatively simple, fast, and cost-effective method that is applicable to both genome and targeted assays. Linked-Reads are generated by performing haplotype-level dilution of long input DNA molecules into >1 million barcoded partitions, generating barcoded short reads within those partitions, and then performing short read sequencing in bulk. We performed 30x Linked-Read genome sequencing on a set of 23 samples with known balanced or unbalanced SVs. Twenty-seven of the 29 known events were detected and another event was called as a candidate. Sequence downsampling was performed on a subset to determine the lowest sequence depth required to detect variations. Copy-number variants can be called with as little as 1-2x sequencing depth (5-10Gb) while balanced events require on the order of 10x coverage for variant calls to be made, although specific signal is clearly present at 1-2x sequencing depth. In addition to detecting a full spectrum of variant types with a single test, Linked-Read sequencing provides base-level resolution of breakpoints, enabling complete resolution of even the most complex chromosomal rearrangements.

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Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach

Parasites & Vectors ◽

10.1186/s13071-021-05009-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Ruoyao Ni ◽

Nian Liu ◽

Mei Li ◽

Weiping Qian ◽

Xinghui Qiu

Keyword(s):

Next Generation Sequencing ◽

Sodium Channel ◽

Phylogenetic Analyses ◽

Cost Effective ◽

Amplicon Sequencing ◽

Anopheles Sinensis ◽

Next Generation ◽

Voltage Gated ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background Anopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China. Methods To understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China. Results Nineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F. Conclusions A cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis. Graphical abstract

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Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases

International Journal of Molecular Sciences ◽

10.3390/ijms22126419 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6419

Author(s):

Janine Reurink ◽

Adrian Dockery ◽

Dominika Oziębło ◽

G. Jane Farrar ◽

Monika Ołdak ◽

...

Keyword(s):

Screening Tool ◽

Usher Syndrome ◽

Copy Number Variants ◽

Genetic Diagnosis ◽

Cost Effective ◽

Single Nucleotide Variants ◽

Effective Screening ◽

Pathogenic Variants ◽

Future Therapy ◽

Molecular Inversion Probe

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

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Next-Generation Molecular Investigations in Lysosomal Diseases: Clinical Integration of a Comprehensive Targeted Panel

Diagnostics ◽

10.3390/diagnostics11020294 ◽

2021 ◽

Vol 11 (2) ◽

pp. 294

Author(s):

Bénédicte Sudrié-Arnaud ◽

Sarah Snanoudj ◽

Ivana Dabaj ◽

Hélène Dranguet ◽

Lenaig Abily-Donval ◽

...

Keyword(s):

Performance Metrics ◽

Age At Onset ◽

Copy Number Variants ◽

Cost Effective ◽

Diagnostic Procedures ◽

Next Generation ◽

Single Nucleotide Variants ◽

Bioinformatics Analyses ◽

Clinical Complications ◽

Lysosomal Disorders

Diagnosis of lysosomal disorders (LDs) may be hampered by their clinical heterogeneity, phenotypic overlap, and variable age at onset. Conventional biological diagnostic procedures are based on a series of sequential investigations and require multiple sampling. Early diagnosis may allow for timely treatment and prevent clinical complications. In order to improve LDs diagnosis, we developed a capture-based next generation sequencing (NGS) panel allowing the detection of single nucleotide variants (SNVs), small insertions and deletions, and copy number variants (CNVs) in 51 genes related to LDs. The design of the LD panel covered at least coding regions, promoter region, and flanking intronic sequences for 51 genes. The validation of this panel consisted in testing 21 well-characterized samples and evaluating analytical and diagnostic performance metrics. Bioinformatics pipelines have been validated for SNVs, indels and CNVs. The clinical output of this panel was tested in five novel cases. This capture-based NGS panel provides an average coverage depth of 474× which allows the detection of SNVs and CNVs in one comprehensive assay. All the targeted regions were covered above the minimum required depth of 30×. To illustrate the clinical utility, five novel cases have been sequenced using this panel and the identified variants have been confirmed using Sanger sequencing or quantitative multiplex PCR of short fluorescent fragments (QMPSF). The application of NGS as first-line approach to analyze suspected LD cases may speed up the identification of alterations in LD-associated genes. NGS approaches combined with bioinformatics analyses, are a useful and cost-effective tool for identifying the causative variations in LDs.

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E-Voting Kepala Desa dan Modal Sosial

Jurnal Sains Komunikasi dan Pengembangan Masyarakat [JSKPM] ◽

10.29244/jskpm.2.6.759-772 ◽

2019 ◽

Vol 2 (6) ◽

pp. 759

Author(s):

Khaulah Afifah ◽

Lala M Kolopaking ◽

Zessy Ardinal Barlan

Keyword(s):

Social Capital ◽

Random Sampling ◽

Qualitative Data ◽

Sampling Technique ◽

Cost Effective ◽

Simple Random Sampling ◽

Community Based ◽

Time Saving ◽

The Social ◽

Voting System

Head of a village election with e-voting system is a new thing for community The success level of e-voting system can be reached by fulfil several principles in order to the implementation going effective and the result of the election can be accepted by all. The objectives of this research is to analyze the relation between the success level of e-voting system with social capital of the community. This research is carried out with the quantitative approach and supported by qualitative data. This research takes 60 respondents using simple random sampling technique. The results showed that the success level of e-voting has a correlation with the level of social capital of the community. Based on the field study, the social capital of the community is classified as high. The high social capital makes the implementation of e-voting successful and the success level is also high, because in the election ten years ago occurred a conflict. The community considers e-voting easier and more practical, cost effective and time-saving, and the results of e-voting are also reliable. A practical and fast of e-voting system can be a solution especially for “rural-urban” community who are busy or work outside the village.Keywords: E-voting, the success level of the system, social capital Pemilihan kepala desa dengan sistem e-voting merupakan hal yang baru bagi masyarakat. Keberhasilan penerapan sistem e-voting dilihat dari terpenuhinya beberapa prinsip agar penerapannya berlangsung efektif dan hasilnya dapat diterima oleh seluruh masyarakat. Penelitian ini bertujuan untuk menganalisis hubungan tingkat keberhasilan sistem e-voting dalam pemilihan kepala desa dengan tingkat modal sosial masyarakat. Bentuk penelitian ini adalah penelitian kuantitatif yang didukung oleh analisis data kualitatif. Penelitian ini mengambil enam puluh responden dengan teknik simple random sampling. Hasil penelitian menunjukkan bahwa tingkat keberhasilan e-voting memiliki hubungan dengan tingkat modal sosial masyarakat. Berdasarkan kajian di lapang, modal sosial masyarakat tergolong tinggi. Tingginya modal sosial tersebut membuat pelaksanaan e-voting berhasil dan tingkat keberhasilannya juga tergolong tinggi karena pada pemilihan sepuluh tahun silam sempat terjadi konflik. Masyarakat menganggap sistem evoting lebih mudah dan praktis, hemat dalam segi biaya dan waktu, serta hasil dari pemilihan juga dapat dipertanggungjawabkan. Sistem e-voting yang praktis dan cepat dapat menjadi solusi khususnya bagi masyarakat daerah “desa-kota” yang memiliki kesibukan atau pekerjaan di luar desa.Kata Kunci: E-voting, keberhasilan sistem, modal sosial.

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Kinetic Behaviour of Amylase According to pH: A New Perspective for Starch Hydrolysis Process

Current Enzyme Inhibition ◽

10.2174/1573408016666200316114808 ◽

2020 ◽

Vol 16 (2) ◽

pp. 135-144

Author(s):

Ravneet K. Grewal ◽

Baldeep Kaur ◽

Gagandeep Kaur

Keyword(s):

Metal Ions ◽

Competitive Inhibition ◽

Deae Cellulose ◽

Kinetic Studies ◽

Cost Effective ◽

Starch Hydrolysis ◽

Divalent Metal Ions ◽

Activity Data ◽

Time Saving ◽

Alkaline Conditions

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency. Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH. Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot. Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5. Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

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Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22041508 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1508

Author(s):

Jordi Maggi ◽

Samuel Koller ◽

Luzy Bähr ◽

Silke Feil ◽

Fatma Kivrak Pfiffner ◽

...

Keyword(s):

Genetic Testing ◽

Long Range ◽

Copy Number Variants ◽

Retinal Disease ◽

Promoter Regions ◽

Long Range Pcr ◽

Cost Efficient ◽

Nucleotide Resolution ◽

Genomic Regions ◽

Next Generation Sequencing Ngs

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

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One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation

Genetics in Medicine ◽

10.1038/s41436-021-01187-w ◽

2021 ◽

Author(s):

Stephen E. Lincoln ◽

Tina Hambuch ◽

Justin M. Zook ◽

Sara L. Bristow ◽

Kathryn Hatchell ◽

...

Keyword(s):

Diagnostic Yield ◽

Copy Number Variants ◽

Low Complexity ◽

Clinical Genetic ◽

Clinical Sensitivity ◽

Pathogenic Variants ◽

Wide Range ◽

Large Indels ◽

Next Generation Sequencing Ngs ◽

The Impact

Abstract Purpose To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. Methods An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)–based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. Results In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. Conclusion The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

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Rare Germline Variants in Chordoma-Related Genes and Chordoma Susceptibility

Cancers ◽

10.3390/cancers13112704 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2704

Author(s):

Sally Yepes ◽

Nirav N. Shah ◽

Jiwei Bai ◽

Hela Koka ◽

Chuzhong Li ◽

...

Keyword(s):

Internal Control ◽

Susceptibility Gene ◽

Copy Number Variants ◽

Bone Cancer ◽

Chinese Patients ◽

European Ancestry ◽

Unknown Etiology ◽

Loss Of Function ◽

Single Nucleotide Variants ◽

Pathogenic Variants

Background: Chordoma is a rare bone cancer with an unknown etiology. TBXT is the only chordoma susceptibility gene identified to date; germline single nucleotide variants and copy number variants in TBXT have been associated with chordoma susceptibility in familial and sporadic chordoma. However, the genetic susceptibility of chordoma remains largely unknown. In this study, we investigated rare germline genetic variants in genes involved in TBXT/chordoma-related signaling pathways and other biological processes in chordoma patients from North America and China. Methods: We identified variants that were very rare in general population and internal control datasets and showed evidence for pathogenicity in 265 genes in a whole exome sequencing (WES) dataset of 138 chordoma patients of European ancestry and in a whole genome sequencing (WGS) dataset of 80 Chinese patients with skull base chordoma. Results: Rare and likely pathogenic variants were identified in 32 of 138 European ancestry patients (23%), including genes that are part of notochord development, PI3K/AKT/mTOR, Sonic Hedgehog, SWI/SNF complex and mesoderm development pathways. Rare pathogenic variants in COL2A1, EXT1, PDK1, LRP2, TBXT and TSC2, among others, were also observed in Chinese patients. Conclusion: We identified several rare loss-of-function and predicted deleterious missense variants in germline DNA from patients with chordoma, which may influence chordoma predisposition and reflect a complex susceptibility, warranting further investigation in large studies.

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Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

Journal of International Medical Research ◽

10.1177/0300060520967778 ◽

2020 ◽

Vol 48 (12) ◽

pp. 030006052096777

Author(s):

Peisong Chen ◽

Xuegao Yu ◽

Hao Huang ◽

Wentao Zeng ◽

Xiaohong He ◽

...

Keyword(s):

Next Generation Sequencing ◽

Ion Torrent ◽

Next Generation ◽

Large Deletions ◽

Different Types ◽

Variant Detection ◽

Polymerase Chain ◽

Next Generation Sequencing Ngs ◽

Accuracy And Repeatability ◽

Generation Sequencing

Introduction To evaluate a next-generation sequencing (NGS) workflow in the screening and diagnosis of thalassemia. Methods In this prospective study, blood samples were obtained from people undergoing genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain reaction (PCR)-amplified and sequenced using the Ion Torrent system and results compared with traditional genetic analyses. Results Of the 359 subjects, 148 (41%) were confirmed to have thalassemia. Variant detection identified 35 different types including the most common. Identification of the mutational sites by NGS were consistent with those identified by Sanger sequencing and Gap-PCR. The sensitivity and specificities of the Ion Torrent NGS were 100%. In a separate test of 16 samples, results were consistent when repeated ten times. Conclusion Our NGS workflow based on the Ion Torrent sequencer was successful in the detection of large deletions and non-deletional defects in thalassemia with high accuracy and repeatability.

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