scholarly journals Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Arteche-López ◽  
A. Ávila-Fernández ◽  
R. Romero ◽  
R. Riveiro-Álvarez ◽  
M. A. López-Martínez ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Turnaround Time ◽  
Copy Number Variations ◽  
Internal Quality ◽  
Single Nucleotide ◽  
Verification Method ◽  
Next Generation Sequencing Ngs ◽  
Improved Accuracy ◽  
Sample Set ◽  
Generation Sequencing

AbstractDespite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

scholarly journals Technologies for Pharmacogenomics: A Review

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1456
Author(s):  
Maaike van der Lee ◽  
Marjolein Kriek ◽  
Henk-Jan Guchelaar ◽  
Jesse J. Swen
Keyword(s):  
Clinical Practice ◽  
Rare Variants ◽  
Turnaround Time ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Long Read ◽  
The Right ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Straightforward Interpretation

The continuous development of new genotyping technologies requires awareness of their potential advantages and limitations concerning utility for pharmacogenomics (PGx). In this review, we provide an overview of technologies that can be applied in PGx research and clinical practice. Most commonly used are single nucleotide variant (SNV) panels which contain a pre-selected panel of genetic variants. SNV panels offer a short turnaround time and straightforward interpretation, making them suitable for clinical practice. However, they are limited in their ability to assess rare and structural variants. Next-generation sequencing (NGS) and long-read sequencing are promising technologies for the field of PGx research. Both NGS and long-read sequencing often provide more data and more options with regard to deciphering structural and rare variants compared to SNV panels—in particular, in regard to the number of variants that can be identified, as well as the option for haplotype phasing. Nonetheless, while useful for research, not all sequencing data can be applied to clinical practice yet. Ultimately, selecting the right technology is not a matter of fact but a matter of choosing the right technique for the right problem.

scholarly journals Different Methods in HPV Genotyping of Anogenital and Oropharyngeal Lesions: Comparison between VisionArray® Technology, Next Generation Sequencing, and Hybrid Capture Assay

2021 ◽  
Vol 2 (1) ◽  
pp. 29-41
Author(s):  
Giorgia Acquaviva ◽  
Michela Visani ◽  
Viviana Sanza ◽  
Antonio De Leo ◽  
Thais Maloberti ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Turnaround Time ◽  
Hpv Infection ◽  
Human Papillomaviruses ◽  
Next Generation ◽  
Hybrid Capture ◽  
Capture Assay ◽  
Anogenital Area ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray® (κ = 0.802), as well as between HC II and VisionArray® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results.

scholarly journals Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elizabeth Jaworski ◽  
Rose M Langsjoen ◽  
Brooke Mitchell ◽  
Barbara Judy ◽  
Patrick Newman ◽  
...  
Keyword(s):  
Full Range ◽  
Virus Genome ◽  
Clinical Samples ◽  
Rna Recombination ◽  
Single Nucleotide ◽  
Reverse Transcription Reaction ◽  
Minority Variants ◽  
Next Generation Sequencing Ngs ◽  
Unique Molecular Identifier ◽  
Generation Sequencing

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

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 Methylmalonic Acidemia with Novel MUT Gene Mutations

2017 ◽  
Vol 2017 ◽  
pp. 1-2
Author(s):  
Inusha Panigrahi ◽  
Savita Bhunwal ◽  
Harish Varma ◽  
Simranjeet Singh
Keyword(s):  
Sanger Sequencing ◽  
Gene Mutations ◽  
Methylmalonic Acidemia ◽  
Feeding Problems ◽  
Novel Variants ◽  
Mut Gene ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Poor Weight Gain

A 5-year-old boy presented with recurrent episodes of fever, feeding problems, lethargy, from the age of 11 months, and poor weight gain. He was admitted and evaluated for metabolic causes and diagnosed as having methylmalonic acidemia (MMA). He was treated with vit B12 and carnitine supplements and has been on follow-up for the last 3 years. Mutation analysis by next generation sequencing (NGS), supplemented with Sanger sequencing, revealed two novel variants in the MUT gene responsible for MMA in exon 5 and exon 3, respectively. Recently he developed dystonic movements including orofacial dyskinesia. With advent of NGS, judicious use of NGS with Sanger sequencing can help identify causative possibly pathogenic mutations.

scholarly journals A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiandong Zeng ◽  
Natalia T. Leach ◽  
Zhaoqing Zhou ◽  
Hui Zhu ◽  
Jean A. Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Causative Mutation ◽  
Next Generation ◽  
Single Nucleotide ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Next Generation Sequencing Ngs ◽  
Reference Bias ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

scholarly journals 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

2021 ◽  
Author(s):  
Gulay Korukluoglu ◽  
Mustafa Kolukirik ◽  
Fatma Bayrakdar ◽  
Gozde Girgin Ozgumus ◽  
Ayse Basak Altas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Clinical Samples ◽  
One Step ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTA one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.

scholarly journals Next-Generation Sequencing Combined With Conventional Sanger Sequencing Reveals High Molecular Diversity in Actinidia Virus 1 Populations From Kiwifruit Grown in China

2020 ◽  
Vol 11 ◽  
Author(s):  
Shaohua Wen ◽  
Guoping Wang ◽  
Zuokun Yang ◽  
Yanxiang Wang ◽  
Min Rao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Molecular Diversity ◽  
Genome Structure ◽  
Viral Disease ◽  
Open Reading Frames ◽  
Next Generation ◽  
Next Generation Sequencing Ngs ◽  
First Time ◽  
Generation Sequencing

Kiwifruit (Actinidia spp.) is native to China. Viral disease–like symptoms are common on kiwifruit plants. In this study, six libraries prepared from total RNA of leaf samples from 69 kiwifruit plants were subjected to next-generation sequencing (NGS). Actinidia virus 1 (AcV-1), a tentative species in the family Closteroviridae, was discovered in the six libraries. Two full-length and two near-full genome sequences of AcV-1 variants were determined by Sanger sequencing. The genome structure of these Chinese AcV-1 variants was identical to that of isolate K75 and consisted of 12 open reading frames (ORFs). Analyses of these sequences together with the NGS-derived contig sequences revealed high molecular diversity in AcV-1 populations, with the highest sequence variation occurring at ORF1a, ORF2, and ORF3, and the available variants clustered into three phylogenetic clades. For the first time, our study revealed different domain compositions in the viral ORF1a and molecular recombination events among AcV-1 variants. Specific reverse transcriptase–polymerase chain reaction assays disclosed the presence of AcV-1 in plants of four kiwifruit species and unknown Actinidia spp. in seven provinces and one city.

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