scholarly journals Measuring Illumina Size Bias Using REcount: A Novel Method for Highly Accurate Quantification of Engineered Genetic Constructs

2018 ◽  
Author(s):  
Daryl M. Gohl ◽  
Aaron Becker ◽  
Darrell Johnson ◽  
Shea Anderson ◽  
Bradley Billstein ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Digital Pcr ◽  
Counting Method ◽  
Novel Method ◽  
Sequencing Platforms ◽  
Next Generation Sequencing Ngs ◽  
Genetic Constructs ◽  
Generation Sequencing ◽  
Accurate Quantification

AbstractQuantification of DNA sequence tags associated with engineered genetic constructs underlies many genomics measurements. Typically, such measurements are done using PCR to enrich sequence tags and add adapters, followed by next-generation sequencing (NGS). However, PCR amplification can introduce significant quantitative error into these measurements. Here we describe REcount, a novel PCR-free direct counting method for NGS-based quantification of engineered genetic constructs. By comparing measurements of defined plasmid pools to droplet digital PCR data, we demonstrate that this method is highly accurate and reproducible. We further demonstrate that the REcount approach is amenable to multiplexing through the use of orthogonal restriction enzymes. Finally, we use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencing platforms.

scholarly journals Current and New Approaches in GMO Detection: Challenges and Solutions

2015 ◽  
Vol 2015 ◽  
pp. 1-22 ◽  
Author(s):  
Marie-Alice Fraiture ◽  
Philippe Herman ◽  
Isabel Taverniers ◽  
Marc De Loose ◽  
Dieter Deforce ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
Digital Pcr ◽  
Detection Methods ◽  
Gmo Detection ◽  
Freedom Of Choice ◽  
Next Generation Sequencing Ngs ◽  
Detection Strategies ◽  
Generation Sequencing ◽  
Accurate Quantification

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review.

scholarly journals Uncovering the Complexity of Transcriptomes with RNA-Seq

2010 ◽  
Vol 2010 ◽  
pp. 1-19 ◽  
Author(s):  
Valerio Costa ◽  
Claudia Angelini ◽  
Italia De Feis ◽  
Alfredo Ciccodicola
Keyword(s):  
Massively Parallel Sequencing ◽  
Point Of View ◽  
Rna Seq ◽  
Specific Expression ◽  
Allele Specific ◽  
Data Files ◽  
Sequencing Platforms ◽  
New Applications ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

In recent years, the introduction of massively parallel sequencing platforms for Next Generation Sequencing (NGS) protocols, able to simultaneously sequence hundred thousand DNA fragments, dramatically changed the landscape of the genetics studies. RNA-Seq for transcriptome studies, Chip-Seq for DNA-proteins interaction, CNV-Seq for large genome nucleotide variations are only some of the intriguing new applications supported by these innovative platforms. Among them RNA-Seq is perhaps the most complex NGS application. Expression levels of specific genes, differential splicing, allele-specific expression of transcripts can be accurately determined by RNA-Seq experiments to address many biological-related issues. All these attributes are not readily achievable from previously widespread hybridization-based or tag sequence-based approaches. However, the unprecedented level of sensitivity and the large amount of available data produced by NGS platforms provide clear advantages as well as new challenges and issues. This technology brings the great power to make several new biological observations and discoveries, it also requires a considerable effort in the development of new bioinformatics tools to deal with these massive data files. The paper aims to give a survey of the RNA-Seq methodology, particularly focusing on the challenges that this application presents both from a biological and a bioinformatics point of view.

Detecting TYMS tandem repeat polymorphism by the PSSD method based on next generation sequecing

2020 ◽  
Vol 15 ◽  
Author(s):  
Binsheng He ◽  
Jialiang Yang ◽  
Geng Tian ◽  
Pingping Bing ◽  
Jidong Lang
Keyword(s):  
Tandem Repeat ◽  
Variable Number ◽  
Read Length ◽  
Line Treatment ◽  
Next Generation ◽  
Gene Encoding ◽  
Sequencing Errors ◽  
Novel Method ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

: Thymidylate synthase (TS) is an important target for folicacid inhibitors such as pemetrexed, which has considerable effects on the first-line treatment, second-line treatment and maintenance therapy for patients with late-stage non-small cell lung cancer (NSCLC). Therefore, detecting mutations in TYMS gene encoding TS is critical in clinical applications. With the development of the next-generation sequencing (NGS) technology, the accuracy of TYMS mutation detection is getting higher and higher. However, traditional methods suffer from false-positives and false-negatives caused by factors like limited sequencing read length and sequencing errors. In this study, we have developed a novel method based on "paired seed sequence distance” (PSSD) to detect the variable number of tandem repeat (VNTR) mutation for TYMS. Our method not only improves the detection rate and accuracy of TYMS VNTR mutations, but also avoids problems caused by sequencing errors and limited sequencing length. This method provides a new solution for similar polymorphism analyses and other sequencing analyses.

scholarly journals Analytical Performance of NGS-Based Molecular Genetic Tests Used in the Diagnostic Workflow of Pheochromocytoma/Paraganglioma

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4219
Author(s):  
Balazs Sarkadi ◽  
Istvan Liko ◽  
Gabor Nyiro ◽  
Peter Igaz ◽  
Henriett Butz ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Molecular Genetic ◽  
Cost Effective ◽  
Gene Panel ◽  
Diagnostic Tools ◽  
Target Capture ◽  
Whole Exome ◽  
Sequencing Platforms ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next Generation Sequencing (NGS)-based methods are high-throughput and cost-effective molecular genetic diagnostic tools. Targeted gene panel and whole exome sequencing (WES) are applied in clinical practice for assessing mutations of pheochromocytoma/paraganglioma (PPGL) associated genes, but the best strategy is debated. Germline mutations of at the least 18 PPGL genes are present in approximately 20–40% of patients, thus molecular genetic testing is recommended in all cases. We aimed to evaluate the analytical and clinical performances of NGS methods for mutation detection of PPGL-associated genes. WES (three different library preparation and bioinformatics workflows) and an in-house, hybridization based gene panel (endocrine-onco-gene-panel- ENDOGENE) was evaluated on 37 (20 WES and 17 ENDOGENE) samples with known variants. After optimization of the bioinformatic workflow, 61 additional samples were tested prospectively. All clinically relevant variants were validated with Sanger sequencing. Target capture of PPGL genes differed markedly between WES platforms and genes tested. All known variants were correctly identified by all methods, but methods of library preparations, sequencing platforms and bioinformatical settings significantly affected the diagnostic accuracy. The ENDOGENE panel identified several pathogenic mutations and unusual genotype–phenotype associations suggesting that the whole panel should be used for identification of genetic susceptibility of PPGL.

scholarly journals A Novel p.G141R Mutation in ILDR1 Leads to Recessive Nonsyndromic Deafness DFNB42 in Two Chinese Han Families

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Xueling Wang ◽  
Longhao Wang ◽  
Hu Peng ◽  
Tao Yang ◽  
Hao Wu
Keyword(s):  
Sanger Sequencing ◽  
Pcr Amplification ◽  
Homozygous Mutation ◽  
Frequent Mutation ◽  
Nonsyndromic Deafness ◽  
Chinese Han ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Genetic hearing impairment is highly heterogeneous. In this study, targeted next-generation sequencing (NGS) in two Chinese Han families identified a novel p.G141R homozygous mutation in ILDR1 as the genetic cause of the deafness. Consistent with the recessive inheritance, cosegregation of the p.G141R variant with the hearing loss was confirmed in members of both families by PCR amplification and Sanger sequencing. SNP genotyping analysis suggested that those two families were not closely related. Our study showed that targeted NGS is an effective tool for diagnosis of genetic deafness and that p.G141R in ILDR1 may be a relatively frequent mutation for DFNB42 in Chinese Hans.

scholarly journals Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

2011 ◽  
Vol 13 ◽  
Author(s):  
Sinuhe Hahn ◽  
Olav Lapaire ◽  
Sevgi Tercanli ◽  
Varaprasad Kolla ◽  
Irene Hösli
Keyword(s):  
Point Mutations ◽  
Digital Pcr ◽  
Maternal Blood ◽  
Noninvasive Detection ◽  
Current Form ◽  
Targeted Ngs ◽  
The Right ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are ‘digital PCR’ and ‘next-generation sequencing’ (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as β-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers.

Phylogeny and reclassification of Carpophilinae (Coleoptera: Nitidulidae), with insights into the origins of anthophily

2020 ◽  
Vol 189 (4) ◽  
pp. 1359-1369
Author(s):  
Gareth S Powell ◽  
Andrew R Cline ◽  
Alexandra G Duffy ◽  
Jennifer M Zaspel
Keyword(s):  
Life Histories ◽  
Ecosystem Functions ◽  
Evolutionary Relationships ◽  
Ancestral State ◽  
Multiple Origins ◽  
The Family ◽  
Novel Method ◽  
Sequencing Platforms ◽  
Generation Sequencing ◽  
High Support

Abstract The family Nitidulidae is known for its diverse life histories, with some species considered economically detrimental pests, while others perform critical ecosystem functions. Despite this, their evolutionary relationships still lack support and understanding. A robust phylogenetic hypothesis and revised taxonomic classification for this group, particularly the subfamily Carpophilinae, could help delineate key lineages for assessing the evolution of both beneficial and pestiferous traits. The most complete molecular phylogeny of Carpophilinae to date, based on 37 ingroup taxa, covering four of the six recognized genera, is presented. Six genetic loci were used (16S, 18S, 28S, COI, H3 and ITS2), providing a total of 3500 bp of data. These data were generated using a novel method discussed here: wide-seq. Wide-seq utilizes next-generation sequencing platforms to generate data for specific loci that overlap with traditional Sanger sequences for the group. Carpophilinae recover as a well-supported clade with respect to the outgroup taxa. The genera Nitops, Urophorus and Caplothorax stat. nov. recover as monophyletic with high support. Caplothorax is formally given generic rank with a redescription and diagnosis. Anthophilous behaviour is coded for all taxa and an ancestral state reconstruction is performed, predicting multiple origins of this behaviour within Carpophilinae.

scholarly journals Applying Next-Generation Sequencing Platforms for Pharmacogenomic Testing in Clinical Practice

2021 ◽  
Vol 12 ◽  
Author(s):  
Alireza Tafazoli ◽  
Henk-Jan Guchelaar ◽  
Wojciech Miltyk ◽  
Adam J. Kretowski ◽  
Jesse J. Swen
Keyword(s):  
Clinical Practice ◽  
Drug Therapy ◽  
Next Generation Sequencing ◽  
Cost Effective ◽  
Genetic Data ◽  
Next Generation ◽  
Cost Effective Approach ◽  
Sequencing Platforms ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Pharmacogenomics (PGx) studies the use of genetic data to optimize drug therapy. Numerous clinical centers have commenced implementing pharmacogenetic tests in clinical routines. Next-generation sequencing (NGS) technologies are emerging as a more comprehensive and time- and cost-effective approach in PGx. This review presents the main considerations for applying NGS in guiding drug treatment in clinical practice. It discusses both the advantages and the challenges of implementing NGS-based tests in PGx. Moreover, the limitations of each NGS platform are revealed, and the solutions for setting up and management of these technologies in clinical practice are addressed.

scholarly journals Environmental DNA (eDNA) reveals endangered narrow sawfish across Indonesian Reefs

2021 ◽  
Vol 944 (1) ◽  
pp. 012020
Author(s):  
L M I Sani ◽  
A K Husna ◽  
B Subhan ◽  
H Madduppa
Keyword(s):  
Filter Paper ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Marine Species ◽  
Visual Census ◽  
Survey Tool ◽  
Polymerase Chain ◽  
Seawater Samples ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Environmental DNA or eDNA is a powerful method to uncover marine organisms in the seawaters. Furthermore, many marine species are difficult to determine in the sea waters because of their rare existence based on the visual census. In this study, we implemented environmental DNA to investigate the presence of the endangered species of narrow sawfish Anoxypristis cuspidata in Indonesia. Four liters of seawater samples were collected at six locations near the coral reefs ecosystem of Indonesia and filtered at 0.45 μm filter paper. DNA was extracted from the filter paper then Polymerase Chain Reaction (PCR) amplification was performed using the cytochrome c oxidase subunit I (COI) primer and analyzed by Next Generation Sequencing (NGS). The findings revealed that narrow sawfish exist in Indonesian waters, and it also simultaneously showed that environmental DNA could detect rare species. The environmental DNA approach to identifying narrow sawfish can provide reliable results and be used as a survey tool to protect endangered threatened and protected (ETP) species.

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