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 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 Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR

BioTechniques ◽  
2020 ◽  
Vol 68 (1) ◽  
pp. 48-51 ◽  
Author(s):  
Christopher R McEvoy ◽  
Timothy Semple ◽  
Bhargavi Yellapu ◽  
David Y Choong ◽  
Huiling Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Limiting Factor ◽  
Next Generation ◽  
Tumor Biopsy ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

scholarly journals One byte at a time: evidencing the quality of clinical service next-generation sequencing for germline and somatic variants

2019 ◽  
Vol 28 (2) ◽  
pp. 202-212 ◽  
Author(s):  
Maria Weronika Gutowska-Ding ◽  
Zandra C. Deans ◽  
Christophe Roos ◽  
Jukka Matilainen ◽  
Farrah Khawaja ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Molecular Techniques ◽  
Performance Criteria ◽  
Next Generation ◽  
Gene Target ◽  
Clinical Diagnostic ◽  
And Performance ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is replacing other molecular techniques to become the de facto gene diagnostics approach, transforming the speed of diagnosis for patients and expanding opportunities for precision medicine. Consequently, for accredited laboratories as well as those seeking accreditation, both objective measures of quality and external review of laboratory processes are required. External quality assessment (EQA), or Proficiency Testing (PT), can assess a laboratory’s service through an independent external agency, the EQA provider. The analysis of a growing number of genes and whole exome and genomes is now routine; therefore, an EQA must be delivered to enable all testing laboratories to participate. In this paper, we describe the development of a unique platform and gene target independent EQA scheme for NGS, designed to scale from current to future requirements of clinical diagnostic laboratories testing for germline and somatic variants. The EQA results from three annual rounds indicate that clinical diagnostic laboratories are providing an increasingly high-quality NGS service and variant calling abilities are improving. From an EQA provider perspective, challenges remain regarding delivery and performance criteria, as well as in analysing similar NGS approaches between cohorts with meaningful metrics, sample sourcing and data formats.

scholarly journals A Review of Bioinformatics Model and Computational Software of Next Generation Sequencing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jalilah Arijah Mohd Kamarudin ◽  
Afnizanfaizal Abdullah ◽  
Roselina Sallehuddin
Keyword(s):  
Next Generation Sequencing ◽  
Review Paper ◽  
Bioinformatics Analysis ◽  
Variant Calling ◽  
Next Generation ◽  
The Past ◽  
Source Type ◽  
Next Generation Sequencing Ngs ◽  
Future Work ◽  
Generation Sequencing

In the past decade it has become increasingly the effort for researcher to surpass the bioinformatics challenges foremost in next generation sequencing (NGS). This review paper gives an overview of the computational software and bioinformatics model that has been used for next generation sequencing. In this paper, the description on functionalities, source type and website of the program or software are provided. These computational software and bioinformatics model are differentiating into three types of bioinformatics analysis stages including alignment, variant calling and filtering and annotation. Besides, we discuss the future work and the development for new bioinformatics tool to be advanced.

scholarly journals Next-generation Sequence-analysis Toolkit (NeST): A standardized bioinformatics framework for analyzing Single Nucleotide Polymorphisms in next-generation sequencing data

2018 ◽  
Author(s):  
Shashidhar Ravishankar ◽  
Sarah E. Schmedes ◽  
Dhruviben S. Patel ◽  
Mateusz Plucinski ◽  
Venkatachalam Udhayakumar ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Single Nucleotide Polymorphisms ◽  
Variant Calling ◽  
Next Generation Sequencing Data ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Bioinformatics Tools ◽  
Generation Sequencing

AbstractRapid advancements in next-generation sequencing (NGS) technologies have led to the development of numerous bioinformatics tools and pipelines. As these tools vary in their output function and complexity and some are not well-standardized, it is harder to choose a suitable pipeline to identify variants in NGS data. Here, we present NeST (NGS-analysis Toolkit), a modular consensus-based variant calling framework. NeST uses a combination of variant callers to overcome potential biases of an individual method used alone. NeST consists of four modules, that integrate open-source bioinformatics tools, a custom Variant Calling Format (VCF) parser and a summarization utility, that generate high-quality consensus variant calls. NeST was validated using targeted-amplicon deep sequencing data from 245 Plasmodium falciparum isolates to identify single-nucleotide polymorphisms conferring drug resistance. The results were verified using Sanger sequencing data for the same dataset in a supporting publication [28]. NeST offers a user-friendly pipeline for variant calling with standardized outputs and minimal computational demands for easy deployment for use with various organisms and applications.

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