scholarly journals SNP Development in Penaeus vannamei via Next-Generation Sequencing and DNA Pool Sequencing

Fishes ◽  
2021 ◽  
Vol 6 (3) ◽  
pp. 36
Author(s):  
Yongyu Huang ◽  
Lili Zhang ◽  
Hui Ge ◽  
Guodong Wang ◽  
Shiyu Huang ◽  
...  
Keyword(s):  
Population Genetics ◽  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Minor Allele Frequency ◽  
Penaeus Vannamei ◽  
Next Generation ◽  
High Quality ◽  
High Quality Snps ◽  
Dna Pool ◽  
Generation Sequencing

Next-generation sequencing and pool sequencing have been widely used in SNP (single-nucleotide polymorphism) detection and population genetics research; however, there are few reports on SNPs related to the growth of Penaeus vannamei. The purpose of this study was to call SNPs from rapid-growing (RG) and slow-growing (SG) individuals’ transcriptomes and use DNA pool sequencing to assess the reliability of SNPs. Two parameters were applied to detect SNPs. One parameter was the p-values generated using Fisher’s exact test, which were used to calculate the significance of allele frequency differences between RG and SG. The other one was the AFI (minor allele frequency imbalance), which was defined to highlight the fold changes in MAF (minor allele frequency) values between RG and SG. There were 216,015 hypothetical SNPs, which were obtained based on the transcriptome data. Finally, 104 high-quality SNPs and 96,819 low-quality SNPs were predicted. Then, 18 high-quality SNPs and 17 low-quality SNPs were selected to assess the reliability of the detection process. Here, 72.22% (13/18) accuracy was achieved for high-quality SNPs, while only 52.94% (9/17) accuracy was achieved for low-quality SNPs. These SNPs enrich the data for population genetics studies of P. vannamei and may play a role in the development of SNP markers for future breeding studies.

High throughput crop genome genotyping by a combination of pool next generation sequencing and haplotype-based data processing

2021 ◽  
Author(s):  
Michael Schneider ◽  
Asis Shrestha ◽  
Agim Ballvora ◽  
Jens Leon
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Frequency Estimation ◽  
Whole Genome ◽  
Next Generation ◽  
Conservation Genomics ◽  
High Coverage ◽  
Allele Frequency Estimation ◽  
Low Coverage ◽  
Generation Sequencing

Abstract BackgroundThe identification of environmentally specific alleles and the observation of evolutional processes is a goal of conservation genomics. By generational changes of allele frequencies in populations, questions regarding effective population size, gene flow, drift, and selection can be addressed. The observation of such effects often is a trade-off of costs and resolution, when a decent sample of genotypes should be genotyped for many loci. Pool genotyping approaches can derive a high resolution and precision in allele frequency estimation, when high coverage sequencing is utilized. Still, pool high coverage pool sequencing of big genomes comes along with high costs.ResultsHere we present a reliable method to estimate a barley population’s allele frequency at low coverage sequencing. Three hundred genotypes were sampled from a barley backcross population to estimate the entire population’s allele frequency. The allele frequency estimation accuracy and yield were compared for three next generation sequencing methods. To reveal accurate allele frequency estimates on a low coverage sequencing level, a haplotyping approach was performed. Low coverage allele frequency of positional connected single polymorphisms were aggregated to a single haplotype allele frequency, resulting in two to 271 times higher depth and increased precision. We compared different haplotyping tactics, showing that gene and chip marker-based haplotypes perform on par or better than simple contig haplotype windows. The comparison of multiple pool samples and the referencing against an individual sequencing approach revealed whole genome pool resequencing having the highest correlation to individual genotyping (up to 0.97), while transcriptomics and genotyping by sequencing indicated higher error rates and lower correlations.ConclusionUsing the proposed method allows to identify the allele frequency of populations with high accuracy at low cost. This is particularly interesting for conservation genomics in species with big genomes, like barley or wheat. Whole genome low coverage resequencing at 10x coverage can deliver a highly accurate estimation of the allele frequency, when a loci-based haplotyping approach is applied. Using annotated haplotypes allows to capitalize from biological background and statistical robustness.

scholarly journals Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1691
Author(s):  
Muscarella ◽  
Fabrizio ◽  
De Bonis ◽  
Mancini ◽  
Balsamo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Scale Up ◽  
Cancer Diagnostics ◽  
Sequencing Analysis ◽  
Library Preparation ◽  
Next Generation ◽  
High Quality ◽  
High Coverage ◽  
Dna Recovery ◽  
Generation Sequencing

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

scholarly journals Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing

2019 ◽  
Vol 110 (8) ◽  
pp. 2652-2657 ◽  
Author(s):  
Hidenori Kage ◽  
Shinji Kohsaka ◽  
Aya Shinozaki‐Ushiku ◽  
Yoshihisa Hiraishi ◽  
Jiro Sato ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Lung Tumor ◽  
Next Generation ◽  
High Quality ◽  
Tumor Biopsy ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Molecular epidemiology of RAS/RAF-mutant colorectal cancer metastases.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 470-470
Author(s):  
Chloe Evelyn Atreya ◽  
James Watters ◽  
Steve Rowley ◽  
Joon Sang Lee ◽  
Oleg Iartchouk ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Liver Metastases ◽  
Allele Frequency ◽  
False Positive Rate ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Braf Mutations ◽  
Cancer Metastases ◽  
Formidable Challenge ◽  
Generation Sequencing

470 Background: A comprehensive molecular characterization of primary colorectal cancers (CRC) was recently reported. Less is known about mutation patterns in CRC metastases and association with survival. Our sequencing analysis focused on CRC liver metastases with RAS/RAF mutations, representing a patient population with limited therapeutic options. Methods: DNA was extracted from formalin-fixed paraffin-embedded CRC liver metastases. Fifty tumors found by Sequenom MassARRAY to harbor KRAS, NRAS or BRAF mutations underwent next generation sequencing on the Ion AmpliSeq Comprehensive Cancer Panel of 409 genes. Co-investigators were blinded to Sequenom mutations identified at UCSF. Variants called by Strelka and VarScan were extensively filtered to control the False Positive Rate and find mutations occurring with > 5-10% variant allele frequency compared to normals. The dataset was evaluated for significant co-mutations, biclustering, and population probabilities of mutations. Results: Following sequencing, 37,744 variants were called in 409 genes with a median coverage depth of 1053x. After filtering to minimize false positives, 2335 variants in 315 genes remained. ARID1A and PIK3R1 were the most significantly associated co-mutation pair, P < 3.5e-5. Biclustering showed no stratification of patients; genes stratified only by mutation frequency. Further filtering yielded 1,186 variants present at < 1% allele frequency within 1,000 Genomes, of which 131 variants in 24 genes are referenced in the Catalog of Somatic Mutations in Cancer. In addition to anticipated mutations in mismatch repair genes and the RTK/RAS/PI3K, Wnt, TP53, and TGF beta pathways, infrequent mutations were found in Akt1, mTOR, MET and PPP2R1a. After APC, TP53 was the most commonly mutated gene, in 44% of the tumors (95% cl: 31.1% - 57.8%). Survival was similar with mutation of RAS/RAF plus either TP53 or PIK3CA. Conclusions: Next generation sequencing was used to characterize co-variants in RAS/RAF mutated CRC liver metastases. The complexity of our results is consistent with the clinical observation that targeting RAS/RAF mutated metastatic CRC is a formidable challenge. These analyses may nonetheless inform the design of future clinical trials.

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