scholarly journals Validation of variants using cost effective highresolution melting (HRM) analysis predicted from target re-sequencing in Eucalyptus

2020 ◽  
Vol 79 (2) ◽  
pp. 105-113
Author(s):  
Abdul Bari Muneera Parveen ◽  
Divya Lakshmanan ◽  
Modhumita Ghosh Dasgupta
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Sequence Data ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Time Saving ◽  
Hrm Analysis ◽  
The Cost ◽  
Generation Sequencing

The advent of next-generation sequencing has facilitated large-scale discovery and mapping of genomic variants for high-throughput genotyping. Several research groups working in tree species are presently employing next generation sequencing (NGS) platforms for marker discovery, since it is a cost effective and time saving strategy. However, most trees lack a chromosome level genome map and validation of variants for downstream application becomes obligatory. The cost associated with identifying potential variants from the enormous amount of sequence data is a major limitation. In the present study, high resolution melting (HRM) analysis was optimized for rapid validation of single nucleotide polymorphisms (SNPs), insertions or deletions (InDels) and simple sequence repeats (SSRs) predicted from exome sequencing of parents and hybrids of Eucalyptus tereticornis Sm. ? Eucalyptus grandis Hill ex Maiden generated from controlled hybridization. The cost per data point was less than 0.5 USD, providing great flexibility in terms of cost and sensitivity, when compared to other validation methods. The sensitivity of this technology in variant detection can be extended to other applications including Bar-HRM for species authentication and TILLING for detection of mutants.

scholarly journals Comparison of Normalization Methods for Construction of Large, Multiplex Amplicon Pools for Next-Generation Sequencing

2010 ◽  
Vol 76 (12) ◽  
pp. 3863-3868 ◽  
Author(s):  
J. Kirk Harris ◽  
Jason W. Sahl ◽  
Todd A. Castoe ◽  
Brandie D. Wagner ◽  
David D. Pollock ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Massively Parallel Sequencing ◽  
Sequence Data ◽  
Cost Savings ◽  
Massively Parallel ◽  
Next Generation ◽  
Normalization Methods ◽  
The Cost ◽  
Generation Sequencing

ABSTRACT Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.

scholarly journals Next-Generation Sequencing Technologies in Blood Group Typing

2019 ◽  
Vol 47 (1) ◽  
pp. 4-13 ◽  
Author(s):  
Daniel Fürst ◽  
Chrysanthi Tsamadou ◽  
Christine Neuchel ◽  
Hubert Schrezenmeier ◽  
Joannis Mytilineos ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Blood Group ◽  
Large Scale ◽  
Cost Effective ◽  
Molecular Testing ◽  
Blood Group Antigens ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Blood Group Typing ◽  
Generation Sequencing

Sequencing of the human genome has led to the definition of the genes for most of the relevant blood group systems, and the polymorphisms responsible for most of the clinically relevant blood group antigens are characterized. Molecular blood group typing is used in situations where erythrocytes are not available or where serological testing was inconclusive or not possible due to the lack of antisera. Also, molecular testing may be more cost-effective in certain situations. Molecular typing approaches are mostly based on either PCR with specific primers, DNA hybridization, or DNA sequencing. Particularly the transition of sequencing techniques from Sanger-based sequencing to next-generation sequencing (NGS) technologies has led to exciting new possibilities in blood group genotyping. We describe briefly the currently available NGS platforms and their specifications, depict the genetic background of blood group polymorphisms, and discuss applications for NGS approaches in immunohematology. As an example, we delineate a protocol for large-scale donor blood group screening established and in use at our institution. Furthermore, we discuss technical challenges and limitations as well as the prospect for future developments, including long-read sequencing technologies.

scholarly journals Finding functional disease-associated non-coding variation using next-generation sequencing

2016 ◽  
Author(s):  
Paolo Devanna ◽  
Xiaowei Sylvia Chen ◽  
Joses Ho ◽  
Dario Gajewski ◽  
Alessandro Gialluisi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Binding Sites ◽  
Large Scale ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Next Generation Sequencing Data ◽  
Whole Genome ◽  
Next Generation ◽  
Whole Exome ◽  
Generation Sequencing

ABSTRACTNext generation sequencing has opened the way for the large scale interrogation of cohorts at the whole exome, or whole genome level. Currently, the field largely focuses on potential disease causing variants that fall within coding sequences and that are predicted to cause protein sequence changes, generally discarding non-coding variants. However non-coding DNA makes up ~98% of the genome and contains a range of sequences essential for controlling the expression of protein coding genes. Thus, potentially causative non-coding variation is currently being overlooked. To address this, we have designed an approach to assess variation in one class of non-coding regulatory DNA; the 3′UTRome. Variants in the 3'UTR region of genes are of particular interest because 3'UTRs are responsible for modulating protein expression levels via their interactions with microRNAs. Furthermore they are amenable to large scale analysis as 3′UTR-microRNA interactions are based on complementary base pairing and as such can be predicted in silico at the genome-wide level. We report a strategy for identifying and functionally testing variants in microRNA binding sites within the 3'UTRome and demonstrate the efficacy of this pipeline in a cohort of language impaired children. Using whole exome sequence data from 43 probands, we extracted variants that lay within 3'UTR microRNA binding sites. We identified a common variant (SNP) in a microRNA binding site and found this SNP to be associated with an endophenotype of language impairment (non-word repetition). We showed that this variant disrupted microRNA regulation in cells and was linked to altered gene expression in the brain, suggesting it may represent a risk factor contributing to SLI. This work demonstrates that biologically relevant variants are currently being under-investigated despite the wealth of next-generation sequencing data available and presents a simple strategy for interrogating non-coding regions of the genome. We propose that this strategy should be routinely applied to whole exome and whole genome sequence data in order to broaden our understanding of how non-coding genetic variation underlies complex phenotypes such as neurodevelopmental disorders.

scholarly journals Clinical Use of Next-Generation Sequencing in the Diagnosis of Wilson’s Disease

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Dániel Németh ◽  
Kristóf Árvai ◽  
Péter Horváth ◽  
János Pál Kósa ◽  
Bálint Tobiás ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Wilson’S Disease ◽  
Cost Effective ◽  
Wilson's Disease ◽  
Clinical Use ◽  
Next Generation ◽  
Time Saving ◽  
Cost Effective Method ◽  
Generation Sequencing

Objective. Wilson’s disease is a disorder of copper metabolism which is fatal without treatment. The great number of disease-causingATP7Bgene mutations and the variable clinical presentation of WD may cause a real diagnostic challenge. The emergence of next-generation sequencing provides a time-saving, cost-effective method for full sequencing of the wholeATP7Bgene compared to the traditional Sanger sequencing. This is the first report on the clinical use of NGS to examineATP7Bgene.Materials and Methods. We used Ion Torrent Personal Genome Machine in four heterozygous patients for the identification of the other mutations and also in two patients with no known mutation. One patient with acute on chronic liver failure was a candidate for acute liver transplantation. The results were validated by Sanger sequencing.Results. In each case, the diagnosis of Wilson’s disease was confirmed by identifying the mutations in both alleles within 48 hours. One novel mutation (p.Ala1270Ile) was found beyond the eight other known ones. The rapid detection of the mutations made possible the prompt diagnosis of WD in a patient with acute liver failure.Conclusions. According to our results we found next-generation sequencing a very useful, reliable, time-saving, and cost-effective method for diagnosing Wilson’s disease in selected cases.

scholarly journals Next-Generation Sequencing Techniques for Eukaryotic Microorganisms: Sequencing-Based Solutions to Biological Problems

2010 ◽  
Vol 9 (9) ◽  
pp. 1300-1310 ◽  
Author(s):  
Minou Nowrousian
Keyword(s):  
Next Generation Sequencing ◽  
Protein Interactions ◽  
Large Scale ◽  
De Novo ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Sequencing Data ◽  
Bioinformatics Analyses ◽  
Eukaryotic Microorganisms ◽  
Generation Sequencing

ABSTRACT Over the past 5 years, large-scale sequencing has been revolutionized by the development of several so-called next-generation sequencing (NGS) technologies. These have drastically increased the number of bases obtained per sequencing run while at the same time decreasing the costs per base. Compared to Sanger sequencing, NGS technologies yield shorter read lengths; however, despite this drawback, they have greatly facilitated genome sequencing, first for prokaryotic genomes and within the last year also for eukaryotic ones. This advance was possible due to a concomitant development of software that allows the de novo assembly of draft genomes from large numbers of short reads. In addition, NGS can be used for metagenomics studies as well as for the detection of sequence variations within individual genomes, e.g., single-nucleotide polymorphisms (SNPs), insertions/deletions (indels), or structural variants. Furthermore, NGS technologies have quickly been adopted for other high-throughput studies that were previously performed mostly by hybridization-based methods like microarrays. This includes the use of NGS for transcriptomics (RNA-seq) or the genome-wide analysis of DNA/protein interactions (ChIP-seq). This review provides an overview of NGS technologies that are currently available and the bioinformatics analyses that are necessary to obtain information from the flood of sequencing data as well as applications of NGS to address biological questions in eukaryotic microorganisms.

Researching novel variants involved in Alzheimer’s disease via next generation sequencing variant analysis on RNA sequence data files

2016 ◽  
Vol 5 (01) ◽  
pp. 4748
Author(s):  
Bhagwat R. ◽  
Joshi B. J. ◽  
Chaudhary S. ◽  
Pardeshi T. A.* ◽  
Mujawar S. N. ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Data ◽  
Programming Algorithm ◽  
Future Research ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Nucleotide Polymorphisms ◽  
Next Generation ◽  
Generation Sequencing ◽  
Brain Tissues

Previous and current research studies have been reporting or refuting susceptibility genes which have any minor or major genetic significance to Alzheimer disease. In our study we have obtained datasets from R. N. A. sequencing analysis performed on post-mortem brain tissues of clinically diagnosed Alzheimer’s male patients above the age of 80. The samples included in our study are grouped into three datasets as frontal brain, temporal brain and whole brain. Our sole purpose of the study was to decipher new S. N. PS if any and report any unreported or untested S. N. Ps related to genes. We have performed mRNA sequencing analysis with the help of Next Generation Sequencing GALAXY tool available online. In addition, we have utilized Integrated Genome Browser (IGB) to visualize our results. P. E. RL Programming algorithm was generated and NCBI’s dbSNP (Database of Single Nucleotide Polymorphisms) and Clin V.A. R (Clinical Variation) databases helped to obtain rsids and related information for galaxy analyses results. Our study is reporting and/or confirming pathogenicity of P. R. N. P, A. P. O. E, CST3, TUBB2B and F. T. L in the AD brain tissues. A growing study of PRNP gene has indicated a strong genetic association with AD yet more research with a bigger sample size in different populations will be of certain significance. Further research is needed for studying the TUBB2B gene due to its uncertain significance in the earlier research in Alzheimer’s. F. T. L is a recent gene which has been linked to A. D and future research in AD should be conducted targeting this specific gene.

Utility of Next Generation Sequencing in the Workup and Diagnosis of Myeloproliferative neoplasm in the Peripheral Blood: A single institution experience

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S136-S136
Author(s):  
T Lynn ◽  
A Campbell ◽  
Y Ding
Keyword(s):  
Next Generation Sequencing ◽  
Peripheral Blood ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Cost Effective ◽  
Jak2 V617f ◽  
Next Generation ◽  
Prognostic Information ◽  
Time Saving ◽  
Generation Sequencing

Abstract Introduction/Objective In patients with suspicion of Myeloproliferative neoplasm (MPN) and negative for BCR-ABL1, NCCN guideline currently recommends two molecular workup pathways in peripheral blood: 1) a multi-step reflex mutation testing algorithm including JAK2 V617F, CALR, MPL, JAK2 exon12 or 2) a multigene Next Generation Sequencing (NGS) panel that includes at least JAK2, CALR and MPL genes. Here we report the clinical utilization and impact of a NGS based MPN diagnosis assay. Methods/Case Report Total of 690 consecutive cases at Geisinger between 2019 and 2021 were included in this study. Patient’s CBC showed chronic cytosis in either single or multi-lineage myelopoiesis and was clinically suspicious for MPNs. For BCR-ABL1 negative cases, NGS based MPN diagnostic assay was performed, which include the four disease defining genes recommended by NCCN guideline: JAK2, CALR, MPL, CSF3R as well as three additional genes NRAS, PPM1D and TP53. Variants are classified in to four tiers based on their level of clinical significance. Results (if a Case Study enter NA) Among all cases tested, 25 out of 690 cases (3.6%) were positive for BCR-ABL1 transcript. 20.9% (139 out of 665 BCR-ABL1 negative cases) had at least one variant detected, which included 73 variants in Tier I category (11.0%), 6 variants in Tier II (0.9%), 57 variants in Tier III (8.6%) and 3 variants in Tier IV(0.5%). Among all disease defining mutations, JAK2 V617F was the most commonly detected mutation (59 cases and 8.8%), followed by CALR indel mutations (13 cases and 2.0%). In addition, double variants were detected in total 7 cases (1.0%). Conclusion Comparing to the conventional multi-step sequential workup for MPN diagnosis, the multi-gene NGS panel provides a cost-effective and time- saving solution. When detected concurrently with the disease defining mutations, NRAS, PPM1D and TP53 could provide not only additional prognostic information but also may suggest pending progression in myeloproliferative neoplasms.

Next-Generation Sequencing: An Emerging Tool for Drug Designing

2019 ◽  
Vol 25 (31) ◽  
pp. 3350-3357 ◽  
Author(s):  
Pooja Tripathi ◽  
Jyotsna Singh ◽  
Jonathan A. Lal ◽  
Vijay Tripathi
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Genomic Research ◽  
Biological Research ◽  
Next Generation ◽  
Human Welfare ◽  
Drug Designing ◽  
Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

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