scholarly journals A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms

2020 ◽  
Author(s):  
Matthew L. Bootsma ◽  
Kristen M. Gruenthal ◽  
Garrett J. McKinney ◽  
Levi Simmons ◽  
Loren Miller ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Parentage Analysis ◽  
Model Organisms ◽  
Sander Vitreus ◽  
Target Sequence ◽  
Genetic Stock ◽  
Genetic Stock Identification ◽  
Trade Offs

AbstractTargeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals, but studies describing detailed workflows of GTseq panel development are rare. Here, we develop a dual-purpose GT-seq panel for walleye (Sander vitreus) and discuss trade-offs associated with different development and genotyping approaches. Our GT-seq panel was developed using restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We then conducted simulations to test the utility of loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We conducted three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. Optimization focused on reducing variation in amplification rate among loci and minimizing the proportion of off-target sequence, both of which are important considerations for developing large GT-seq panels. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (1) inexpensive Chelex extractions performed well for genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was likely unnecessary, and (3) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings should aid in their construction in myriad taxa.

scholarly journals In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment

2021 ◽  
Author(s):  
Christoph M. Deeg ◽  
Ben J. G. Sutherland ◽  
Tobi J. Ming ◽  
Colin Wallace ◽  
Kim Jonsen ◽  
...  
Keyword(s):  
Real Time ◽  
Coho Salmon ◽  
Gulf Of Alaska ◽  
Amplicon Sequencing ◽  
Stock Identification ◽  
Nanopore Sequencing ◽  
Genetic Stock ◽  
Genetic Stock Identification ◽  
Batch Sizes ◽  
Sequencing Platforms

Genetic stock identification (GSI) by single nucleotide polymorphism (SNP) sequencing has become the gold standard for stock identification in Pacific salmon, which are found in mixed-stocks during the oceanic phase of their lifecycle. Sequencing platforms currently applied require large batch sizes and multi-day processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third-generation single-molecule sequencing platforms, like the Oxford Nanopore minION, provide base calling on portable, pocket-sized sequencers and hold promise for the application of real-time, in-field stock identification on variable batch sizes. Here we report and evaluate utility and comparability of at-sea stock identification of coho salmon Oncorhynchus kisutch based on targeted SNP amplicon sequencing on the minION platform during the International Year of the Salmon Signature Expedition to the Gulf of Alaska in the winter of 2019. Long read sequencers are not optimized for short amplicons, therefore we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at-sea yielded stock assignment for 50 of the 80 assessed individuals. Nanopore-based SNP calls agreed with Ion Torrent based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed coho salmon in the queried baseline dataset contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time, accuracy, throughput, and cost, as well as augmentation of the existing baselines, specifically in stocks from coastal northern BC and Alaska. If successfully implemented, Nanopore sequencing will provide an alternative method to the large-scale laboratory approach. Genotyping by amplicon sequencing in the hands of diverse stakeholders could inform management decisions over a broad expanse of the coast by allowing the analysis of small batches in remote areas in near real-time.

scholarly journals Population and individual identification of coho salmon in British Columbia through parentage-based tagging and genetic stock identification: an alternative to coded-wire tags

2017 ◽  
Vol 74 (9) ◽  
pp. 1391-1410 ◽  
Author(s):  
Terry D. Beacham ◽  
Colin Wallace ◽  
Cathy MacConnachie ◽  
Kim Jonsen ◽  
Brenda McIntosh ◽  
...  
Keyword(s):  
Coho Salmon ◽  
Direct Sequencing ◽  
Parentage Analysis ◽  
Individual Identification ◽  
Parental Genotype ◽  
Stock Identification ◽  
Nucleotide Polymorphisms ◽  
Genetic Stock ◽  
Genetic Stock Identification ◽  
Assignment Probability

Parentage-based tagging (PBT) and genetic stock identification (GSI) were used to identify individual coho salmon (Oncorhynchus kisutch) to specific populations and brood years. In total, 20 242 individuals from 117 populations were genotyped at 304 single nucleotide polymorphisms (SNPs) via direct sequencing of amplicons. Coho salmon from 15 populations were assigned via parentage analysis that required the genotypes of both parents. The overall accuracy of assignment for 1939 coho salmon to the correct population was 100%, and to correct brood year within population was also 100%. Inclusion of individuals requiring only a single parental genotype for identification resulted in assignments of 2101 individuals, with an accuracy of 99.95% (2000–2001) to population and 100.0% to age. With 23 regions defined by the coded-wire tag (CWT) program, and individuals displaying an assignment probability <0.85 excluded from the analysis, mean regional assignment accuracy of individuals via GSI was 98.4% over all 23 regions. A PBT–GSI or PBT system of identification will provide an alternate method of identification in the assessment and management of Canadian-origin coho salmon relative to the existing CWT program.

scholarly journals dCAPS method: advantages, troubles and solution

2008 ◽  
Vol 53 (No. 9) ◽  
pp. 417-420 ◽  
Author(s):  
M. Hrubá
Keyword(s):  
Genetic Mapping ◽  
Sex Chromosomes ◽  
Restriction Site ◽  
Cost Effective ◽  
Fundamental Principle ◽  
The Other ◽  
Model Organisms ◽  
Target Sequence ◽  
Evolutionary Studies ◽  
Sequence Polymorphisms

In our work, we focus on the evolutionary studies of sex chromosomes. As model organisms we use several species of the plant genus <i>Silene</i>. An important part of our research is represented by genetic mapping based on the assays of DNA length or sequence polymorphisms. Apart from the other methods we also use the dCAPS method, which is very useful for detection of the sequence polymorphisms (SNPs). This method is unique as it is able to detect SNPs that are not situated in any restriction site; a fundamental principle of this method is usage of primer designed with one or two mismatches that bring into the target sequence the mutation in vicinity of SNP. Using this method, we found out some improvements that can make analyses more cost-effective.

Comprehensive microsatellite baseline for genetic stock identification of Atlantic salmon (Salmo salar L.) in northernmost Europe

2017 ◽  
Vol 74 (8) ◽  
pp. 2159-2169 ◽  
Author(s):  
Mikhail Ozerov ◽  
Juha-Pekka Vähä ◽  
Vidar Wennevik ◽  
Eero Niemelä ◽  
Martin-A. Svenning ◽  
...  
Keyword(s):  
Atlantic Salmon ◽  
Salmo Salar ◽  
Stock Identification ◽  
Genetic Stock ◽  
Atlantic Salmon Salmo Salar ◽  
Genetic Stock Identification

An International Campaign for Agricultural and Livestock Genomics (CALG)

2002 ◽  
Vol 06 (24) ◽  
pp. 958-965
Author(s):  
Jun Yu ◽  
Jian Wang ◽  
Huanming Yang
Keyword(s):  
Large Scale ◽  
Cost Effective ◽  
Model Organisms ◽  
Environmental Biology ◽  
Cdna Sequences ◽  
Governmental Agencies ◽  
Technology Innovations ◽  
A Genome ◽  
Starting Point ◽  
The Cost

A coordinated international effort to sequence agricultural and livestock genomes has come to its time. While human genome and genomes of many model organisms (related to human health and basic biological interests) have been sequenced or plugged in the sequencing pipelines, agronomically important crop and livestock genomes have not been given high enough priority. Although we are facing many challenges in policy-making, grant funding, regional task emphasis, research community consensus and technology innovations, many initiatives are being announced and formulated based on the cost-effective and large-scale sequencing procedure, known as whole genome shotgun (WGS) sequencing that produces draft sequences covering a genome from 95 percent to 99 percent. Identified genes from such draft sequences, coupled with other resources, such as molecular markers, large-insert clones and cDNA sequences, provide ample information and tools to further our knowledge in agricultural and environmental biology in the genome era that just comes to its accelerated period. If the campaign succeeds, molecular biologists, geneticists and field biologists from all countries, rich or poor, would be brought to the same starting point and expect another astronomical increase of basic genomic information, ready to convert effectively into knowledge that will ultimately change our lives and environment into a greater and better future. We call upon national and international governmental agencies and organizations as well as research foundations to support this unprecedented movement.

scholarly journals Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

2021 ◽  
Author(s):  
Seppo Virtanen ◽  
Schahzad Saqib ◽  
Tinja Kanerva ◽  
Pekka Nieminen ◽  
Ilkka Kalliala ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Extensive Literature ◽  
Metagenomic Sequencing ◽  
Text Extraction ◽  
Healthy Women ◽  
Bacteria And Fungi

Abstract Background: Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial populations. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS-1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing. Results: Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment guided tool provided ecosystem specific annotations that were confirmed by Metagenomic Phylogenetic Analysis (MetaPhlAn). The metagenome analysis revealed distinct functional differences between the bacterial community types while fungi were undetected, despite being identified in all samples based on ITS amplicons. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.Conclusion: Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Text extraction-based annotation tool facilitates the characterization and interpretation of defined microbial communities from rapidly accumulating sequencing and metadata readily available through public databases.

scholarly journals A practical guide to buildde-novoassemblies for single tissues of non-model organisms: the example of a Neotropical frog

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3702 ◽  
Author(s):  
Santiago Montero-Mendieta ◽  
Manfred Grabherr ◽  
Henrik Lantz ◽  
Ignacio De la Riva ◽  
Jennifer A. Leonard ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Cost Effective ◽  
Model Organisms ◽  
Rna Seq ◽  
Assembly Pipeline ◽  
Wide Variability ◽  
History Of ◽  
Inexperienced User

Whole genome sequencing (WGS) is a very valuable resource to understand the evolutionary history of poorly known species. However, in organisms with large genomes, as most amphibians, WGS is still excessively challenging and transcriptome sequencing (RNA-seq) represents a cost-effective tool to explore genome-wide variability. Non-model organisms do not usually have a reference genome and the transcriptome must be assembledde-novo. We used RNA-seq to obtain the transcriptomic profile forOreobates cruralis, a poorly known South American direct-developing frog. In total, 550,871 transcripts were assembled, corresponding to 422,999 putative genes. Of those, we identified 23,500, 37,349, 38,120 and 45,885 genes present in the Pfam, EggNOG, KEGG and GO databases, respectively. Interestingly, our results suggested that genes related to immune system and defense mechanisms are abundant in the transcriptome ofO. cruralis. We also present a pipeline to assist with pre-processing, assembling, evaluating and functionally annotating ade-novotranscriptome from RNA-seq data of non-model organisms. Our pipeline guides the inexperienced user in an intuitive way through all the necessary steps to buildde-novotranscriptome assemblies using readily available software and is freely available at:https://github.com/biomendi/TRANSCRIPTOME-ASSEMBLY-PIPELINE/wiki.

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