Single Nucleotide Variant Detection Using Next Generation Sequencing

2015 ◽  
pp. 109-127 ◽  
Author(s):  
David H. Spencer ◽  
Bin Zhang ◽  
John Pfeifer
Keyword(s):  
Next Generation Sequencing ◽  
Single Nucleotide Variant ◽  
Next Generation ◽  
Single Nucleotide ◽  
Variant Detection ◽  
Generation Sequencing

scholarly journals Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

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.

scholarly journals Targeted next generation sequencing with an extended gene panel does not impact variant detection in mitochondrial diseases

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Morgane Plutino ◽  
Annabelle Chaussenot ◽  
Cécile Rouzier ◽  
Samira Ait-El-Mkadem ◽  
Konstantina Fragaki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mitochondrial Diseases ◽  
Gene Panel ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Variant Detection ◽  
Generation Sequencing

scholarly journals Repli-seq: genome-wide analysis of replication timing by next-generation sequencing

2017 ◽  
Author(s):  
Claire Marchal ◽  
Takayo Sasaki ◽  
Daniel Vera ◽  
Korey Wilson ◽  
Jiao Sima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Replication Timing ◽  
Nucleotide Polymorphisms ◽  
Robust Methods ◽  
Next Generation ◽  
Single Nucleotide ◽  
Cellular Processes ◽  
Genome Wide ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTCycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.

scholarly journals A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing

2016 ◽  
Vol 140 (10) ◽  
pp. 1085-1091 ◽  
Author(s):  
Eric J. Duncavage ◽  
Haley J. Abel ◽  
Jason D. Merker ◽  
John B. Bodner ◽  
Qin Zhao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
In Silico ◽  
Absolute Difference ◽  
Ion Torrent ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Clinical Laboratories ◽  
Generation Sequencing

Context.—Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. Objective.—To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

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