scholarly journals A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP-resistant enzymatic digestion

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Peng Peng ◽  
Yanjuan Xu ◽  
Adrian M. Di Bisceglie ◽  
Xiaofeng Fan
Keyword(s):  
Next Generation Sequencing ◽  
Restriction Enzymes ◽  
Enzymatic Digestion ◽  
Target Enrichment ◽  
Next Generation ◽  
Two Phase ◽  
Low Efficiency ◽  
Viral Sequencing ◽  
Enrichment Strategy ◽  
Generation Sequencing

Abstract Objective Owing to the overwhelming dominance of human and commensal microbe sequences, low efficiency is a major concern in clinical viral sequencing using next-generation sequencing. DNA composed of 7-deaza-2′-deoxyguanosine 5′-triphosphate (c7dGTP), an analog of deoxyguanosine triphosphate (dGTP), is resistant to selective restriction enzymes. This characteristic has been utilized to develop a novel strategy for target enrichment in next-generation sequencing. Results The new enrichment strategy is named target enrichment via enzymatic digestion in next-generation sequencing (TEEDseq). It combined 7-deaza-2′-deoxyguanosine 5′-triphosphate (c7dGTP)-involved primer extension, splinter-assisted intracellular cyclization, c7dGTP)-resistant enzymatic digestion, and two-phase rolling cycle amplification. We first estimated c7dGTP for its efficiency in PCR amplification and its resistance to three restriction enzymes, AluI, HaeIII, and HpyCH4V. We then evaluated TEEDseq using a serum sample spiked with a 1311-bp hepatitis B virus (HBV) fragment. TEEDseq achieved an HBV on-target rate of 3.31 ± 0.39%, which was equivalent to 454× the enrichment of direct Illumina sequencing. Therefore, the current study has provided a concept proof for TEEDseq as an alternative option for clinical viral sequencing that requires an enrichment in next-generation sequencing.

Advances in clinical next-generation sequencing: target enrichment and sequencing technologies

2016 ◽  
Vol 16 (3) ◽  
pp. 357-372 ◽  
Author(s):  
Leomar Y. Ballester ◽  
Rajyalakshmi Luthra ◽  
Rashmi Kanagal-Shamanna ◽  
Rajesh R. Singh
Keyword(s):  
Next Generation Sequencing ◽  
Target Enrichment ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Generation Sequencing

scholarly journals Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jin Sun No ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Seung-Ho Lee ◽  
Jeong-Ah Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome ◽  
Target Enrichment ◽  
Next Generation ◽  
Apodemus Agrarius ◽  
Target Capture ◽  
Generation Sequencing

Abstract Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

Target enrichment using capture probes: The future of HLA typing by next generation sequencing?

2015 ◽  
Vol 76 ◽  
pp. 165
Author(s):  
Hayley Hogan ◽  
Rhys Cransberg ◽  
Megan Jordan ◽  
Damian Goodridge ◽  
David Sayer
Keyword(s):  
Next Generation Sequencing ◽  
Hla Typing ◽  
Target Enrichment ◽  
Next Generation ◽  
The Future ◽  
Generation Sequencing

scholarly journals The use of next-generation sequencing in clinical diagnosis of familial hypercholesterolemia

2013 ◽  
Vol 15 (12) ◽  
pp. 948-957 ◽  
Author(s):  
Jana Vandrovcova ◽  
Ellen R.A. Thomas ◽  
Santosh S Atanur ◽  
Penny J. Norsworthy ◽  
Clare Neuwirth ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Familial Hypercholesterolemia ◽  
Molecular Diagnosis ◽  
Genetic Diagnosis ◽  
Target Enrichment ◽  
Next Generation ◽  
Large Deletions ◽  
Number Of Patients ◽  
The Cost ◽  
Generation Sequencing

Abstract Purpose: Familial hypercholesterolemia is a common Mendelian disorder associated with early-onset coronary heart disease that can be treated by cholesterol-lowering drugs. The majority of cases in the United Kingdom are currently without a molecular diagnosis, which is partly due to the cost and time associated with standard screening techniques. The main purpose of this study was to test the sensitivity and specificity of two next-generation sequencing protocols for genetic diagnosis of familial hypercholesterolemia. Methods: Libraries were prepared for next-generation sequencing by two target enrichment protocols; one using the SureSelect Target Enrichment System and the other using the PCR-based Access Array platform. Results: In the validation cohort, both protocols showed 100% specificity, whereas the sensitivity for short variant detection was 100% for the SureSelect Target Enrichment and 98% for the Access Array protocol. Large deletions/duplications were only detected using the SureSelect Target Enrichment protocol. In the prospective cohort, the mutation detection rate using the Access Array was highest in patients with clinically definite familial hypercholesterolemia (67%), followed by patients with possible familial hypercholesterolemia (26%). Conclusion: We have shown the potential of target enrichment methods combined with next-generation sequencing for molecular diagnosis of familial hypercholesterolemia. Adopting these assays for patients with suspected familial hypercholesterolemia could improve cost-effectiveness and increase the overall number of patients with a molecular diagnosis. Genet Med 15 12, 948–957.

scholarly journals Comparison of Commercially Available Target Enrichment Methods for Next-Generation Sequencing

2013 ◽  
Vol 24 (2) ◽  
pp. 73-86 ◽  
Author(s):  
K. Bodi ◽  
A. G. Perera ◽  
P. S. Adams ◽  
D. Bintzler ◽  
K. Dewar ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Target Enrichment ◽  
Next Generation ◽  
Generation Sequencing ◽  
Enrichment Methods

scholarly journals A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ulrike Frank ◽  
Susanne Kublik ◽  
Dörte Mayer ◽  
Marion Engel ◽  
Michael Schloter ◽  
...  
Keyword(s):  
Cell Death ◽  
Next Generation Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Target Enrichment ◽  
Next Generation ◽  
Mutant Screen ◽  
Specific Target ◽  
Dna Insertion ◽  
Generation Sequencing

Abstract Background Nitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death. Results Altogether 14,282 lines of SALK T-DNA insertion mutants were screened. Growing 1000 pooled mutant lines per tray and simultaneous NO2 fumigation of 4 trays in parallel facilitated high-throughput screening. Candidate mutants were selected based on visible symptoms. Sensitive mutants showed lesions already after fumigation for 1 h with 10 ppm (ppm) NO2 whereas tolerant mutants were hardly damaged even after treatment with 30 ppm NO2. Identification of T-DNA insertion sites by adapter ligation-mediated PCR turned out to be successful but rather time consuming. Therefore, next generation sequencing after T-DNA-specific target enrichment was tested as an alternative screening method. The targeted genome sequencing was highly efficient due to (1.) combination of the pooled DNA from 124 candidate mutants in only two libraries, (2.) successful target enrichment using T-DNA border-specific 70mer probes, and (3.) stringent filtering of the sequencing reads. Seventy mutated genes were identified by at least 3 sequencing reads. Ten corresponding mutants were re-screened of which 8 mutants exhibited NO2-sensitivity or -tolerance confirming that the screen yielded reliable results. Identified candidate genes had published functions in HR, pathogen resistance, and stomata regulation. Conclusions The presented NO2 dead-or-alive screen combined with next-generation sequencing after T-DNA-specific target enrichment was highly efficient. Two researchers finished the screen within 3 months. Moreover, the target enrichment approach was cost-saving because of the limited number of DNA libraries and sequencing runs required. The experimental design can be easily adapted to other screening approaches e.g. involving high-throughput treatments with abiotic stressors or phytohormones.

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