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View our PAG 2019 workshop presentations and scientific posters

Amazing Aquaculture, Better Breeding & Superior Seeds: Partnering for Accelerating Agrigenomics

Applications of genomic technologies are driving faster agriculture advancements and rapid, data-driven research and development. During the workshop, four researchers will discuss applications of genomic technologies for diverse applications ranging from using NGS for advanced sunflower breeding and developing superior soybeans to application of microarrays for breeding of marine aquaculture through applied genomics.  Thermo Fisher Scientific provides innovative tools and solutions to accelerate plant and animal research.

Workshop
Jan 12, 2019 - Jan 16, 2019

Farhad Ghavami, PhD

Advancement in sunflower breeding using an optimal genotyping-by-sequencing AgriSeq panel designed for QTL mapping and genomic selection

Farhad Ghavami, PhD
Chief Scientific Officer (CSO), Agrigenomics
Eurofins BioDiagnostics Inc.
1:00–1:30 p.m.
The advent of next-generation sequencing technologies and the ongoing reduction of sequencing costs have made genotyping-by-sequencing (GBS) a common practice for genotyping in plant and animal breeding. Among different methods of GBS, amplicon-based GBS is an accurate and economical method that can expedite any QTL mapping projects and genomic selection programs.

The first sunflower 10K SNP array was developed through a collaboration between Eurofins BioDiagnostics and the National Sunflower Association (NSA). The array was used on three mapping populations, and a consensus map was created consisting of 5,019 SNP and 118 SSR markers distributed on all 17 linkage groups.

Since the development of the chip in 2012, more than 500 varieties have been genotyped using the NSA array. In 2017 the best markers with optimal performance and high minor allele frequency (MAF) were selected to develop an Applied Biosystems AgriSeq GBS panel. An AgriSeq panel containing 700 of these markers well distributed on the genome has been created, which can help sunflower researchers with QTL mapping, marker-assisted backcrossing, and genomic selection projects.


Farhad Ghavami, PhD

Use of the Axiom and Eureka genotyping platforms to fill a gap in low to medium density genotyping

Jason Nichols
Senior Team Leader, Molecular Analytics
Syngenta Crop Protection, LLC
1:30–2:00 p.m.
The application of genotypic data to support molecular breeding of commercial crop species faces myriad challenges including cost pressure, demands for ever shorter turn-around times, species and genome complexity, etc. Recently, Syngenta has adopted the Applied Biosystems Axiom 384 HT Array platform for the majority of our crops’ needs for medium density fingerprinting. In parallel, and for select crops and applications where a lower density, more agile platform is required, we have also implemented the Applied Biosystems Eureka Genotyping platform. The combination of these two platforms fills a gap in our ability to generate low to medium density genotypic data at large scale, and offers opportunities to incorporate Copy Number Variation and Presence/Absence genetic markers into a genotyping workflow. We will present examples of genotyping applications that are leveraging these platforms, and share some learnings and best practices based upon our experience to date.


Gaspar Malone, PhD

AgriSeq technology: T-GBS as a potential tool for molecular breeding approaches—a practical overview by GDM Seeds

Gaspar Malone, PhD
Biotechnology Research Manager
GDM Seeds
2:00-2:30 p.m.
Molecular markers have allowed breeding companies to enhance the plant selection accuracy year by year. However, a high cost and restricted throughput of some technologies have become a challenge to meet the need of companies. Targeting genotyping-by-sequencing (T-GBS) has appeared as a good alternative, proving to be a low cost and ultra–high-throughput method while allowing breeding companies to optimize their breeding selection pipelines to help reduce time and cost.

GDM Seeds is an Argentinian breeding company with research stations in Argentina, Brazil, and the US. Since 2016, through a partnership with Thermo Fisher Scientific, GDM has expanded its technological portfolio with the first Ion S5 XL platform (now Ion GeneStudio Prime) in Brazil, with the goal of accelerating the molecular breeding selection program.

T-GBS is allowing GDM Seeds to explore new molecular breeding tools such as genome-wide selection, background recovery, and multitrait marker selection, previously unavailable to be explored by genotyping platforms based on “one-by-one” analysis. Ion S5 XL platform and AgriSeq technology are coming to revolutionize marker-assisted selection for crop breeding companies, offering an ultra–high-throughput genotyping output at a relatively low cost per sample. Several hundred samples can be genotyped with several thousands of markers (up to 5,000 SNPs) in some hours.

At GDM Seeds, panels of 1,500 and 3,000 SNPs can be analyzed in up to 1,536 samples per sequencing run, in less than 24 hours. That means more than 4.5 million data points per day or more than 12 million data points per week.


Professor Victor Martinez Moncada, PhD

Diversification of marine aquaculture through genomics: applications to breeding programs in novel species

Professor Victor Martinez Moncada, PhD
Director
FAVET-INBIOGEN, Universidad de Chile
2:30-3:00 p.m.
Many countries are actively investing in the development of new species in marine aquaculture because this sector offers considerable potential to address food security and growing demands in consumer choice. In Chile, there is significant interest in the use of endemic species to diversify the existing industry, which in turn supports a significant component of the economy. However, new breeding programs based on wild broodstock are challenged by lack of knowledge on trait variation and breeding values, extent of population structure and local adaptation in source populations, and on breeding systems. The latter in particular is problematic, because controlled crosses are not feasible in species that rely on communal spawning behaviors. Therefore, the relationship between effective and census population sizes is often unknown. This lack of knowledge hinders accurate assessment of relatedness and in-breeding, and limits genetic gains.

Here, we describe the development and use of genomics tools for the rapid development of sound breeding programs in the kingfish Seriola lalandii, a species of high interest to the seafood industry. We first developed sex-specific genome assemblies from 50 fully sequenced individuals, and used genome-wide association studies and haplotype diversity measures to identify the locus underlying sex determination. Allele-specific gene expression derived from transcriptomic data of developing larvae was used to pinpoint causal loci involved in deformities. The transcriptome data was also used to annotate the genome.

Estimates of genetic diversity and population structure in putative founder broodstock collected in fisheries were obtained using an Applied Biosystems Axiom 90K SNP array that was developed as part of our efforts. We found high levels of individual in-breeding in some founders, but the overall haplotype diversity indicated a high effective population size in the founders. The extent of linkage disequilibrium was efficiently captured by the array, given the genome size. We identified the likely sex ratio in communal breeding in captivity, revealing the contribution of one or two individuals in any spawning event. This information informed breeding protocols going forward, and will also aid breeding programs for other species of the genera.

Finally, deterministic simulations have been used to demonstrate the feasibility of genome selection to improve genetic gains. The information, tools and techniques are being incorporated into breeding programs aimed at increasing the profitability of Seriola production and adding to the diversification of Chilean aquaculture.


Scientific posters

Angela Burrell, Prasad Siddavatam, Michelle Swimley, Chris Willis, Haktan Suren, Krishna Gujjula and Rick Conrad

Parentage testing and genomics-assisted breeding are critical aspects of successful veterinary management. Due to its highly accurate and reproducible results, targeted GBS is becoming an increasingly favored technology for SNP genotyping. With the utilization of next-generation sequencing, labs can test hundreds of samples across thousands of SNPs simultaneously in a simple high-throughput workflow starting from either extracted nucleic acid or crude lysis samples.

We developed two targeted sequencing panels, one for canine parentage verification and one for canine genetic defect/trait identification. Utilizing the Applied Biosystems AgriSeq HTS Library Kit, a high-throughput targeted amplification and re-sequencing workflow, each panel’s performance was tested on >96 diverse DNA samples. Libraries were sequenced on the Ion S5 system using an Ion 540 chip with genotyping calling generated using the Torrent Variant Caller (TVC) plugin.

The mean genotype call rate of markers across the samples was >95% for both panels. Concordance across replicate library preparations and independent sequencing runs was >99% for both panels. Each panel’s results were compared with results
from an Applied Biosystems Axiom Canine HD Array, qPCR, and/or CE sequencing for orthogonal confirmation of genotype accuracy, and the genotype calls were >99% concordant with the AgriSeq workflows. 

The data demonstrates the utility of the AgriSeq targeted GBS approach for canine SNP genotyping applications.

For Research Use Only. Not for use in diagnostic procedures

Jason Wall, Rick Conrad, Claudio Carrasco, Prasad Siddavatm and Christopher C. Adams

Attractive and valuable high-throughput genotyping solutions for parentage and breeding applications require the ability to simultaneously interrogate hundreds to thousands of genetic loci both easily and economically. One disadvantage of many high-throughput genotyping technologies is the lengthy lead times and considerable cost associated with changing the genomic marker content (targeted loci) in any particular assay. The AgriSeq targeted genotyping-by-sequencing (GBS) solution for plant and animal genotyping does not suffer from this problem because the technology relies on a pool of PCR oligonucleotides that can be quickly, easily and inexpensively changed to accommodate always improving knowledge of genomic function. If and when the need arises to alter the content of a marker panel all that is required is the design and synthesis of additional PCR primers, which are then simply spiked into existing assay pools. In addition, AgriSeq genotyping panels can be ordered in plate format in which primer pairs for marker-containing amplicons are individually aliquoted, helping to enable the user to drop unneeded amplicons or re-formulate primer pools (panels) in any combination desired.

Furthermore, individual panels targeting specific species can be mixed together, creating a multi-species panel, while still helping to enable species-specific genotyping. For example, a mixture of three mid-density panels for dog, cat and horse not only allowed for accurate species-specific genotyping, but also enabled the accurate assignment of species to unknown gDNA samples being tested. This unparalleled flexibility in a highly multiplexed genotyping platform provides users unlimited avenues for customizing their genotyping workflows.

For Research Use Only. Not for use in diagnostic procedures.

Ali Pirani, Joseph Foster, Alessandro Davassi, Shantanu Kaushikkar, Brant Wong, Mohini Patil and Luis Jevons

The high-throughput agricultural genotyping landscape encompasses a broad range of applications and technical platforms. One of the major challenges to adopting a new platform or performing meta-analyses is data format congruity. Biallelic genotypes are recorded in one of three ways: “AA”, “AB” and “BB” call codes, “0”, “1”, and “2” numeric call codes and base calls “A”, “T”, “G” or “C”. For call codes and numeric call codes, the A and B alleles must be designated. Historically, two formats have dominated the designation of variant alleles: “Forward” and “TOP”. For biallelic SNPs this can create a situation where the “A” allele designated by one format differs from the other.

To support cross-platform, high-throughput genotyping analysis, Thermo Fisher Scientific has developed the Applied Biosystems Axiom Long Format Export (AxLE) Tool. The tool converts Axiom genotype data to the “TOP” format and converts the output into a format that is similar to the long format options for other platforms. This makes Axiom genotyping easier to integrate with existing downstream analysis pipelines and large scale meta-analyses of several cross-platform datasets.

A clear requirement for the standardization of allele designation is in the application of genotyping data to genetic evaluation systems. To support this specific use case in dairy cattle, Thermo Fisher Scientific has developed the Applied Biosystems Council on Dairy Cattle Breeding (CDCB) Export Tool, which generates a format that enables direct upload to the CDCB website. This free tool can be utilized on any custom or catalog Applied Biosystems Axiom bovine array.

For Research Use Only. Not for use in diagnostic procedures.

Prasad Siddavatam, Haktan Suren, Krishna Reddy Gujjula and Jeanette Schmidt

Traditionally, high-throughput genotyping has been carried out by array-based technologies.

AgriSeq genotyping-by-sequencing (GBS) with Ion Torrent sequencing technology offers a faster, flexible, multiplexing, customizable, cost-effective alternative solution to study 50 to 5,000 markers. AgriSeq GBS also provides capability to multiplex up to 1,536 samples in a single sequencing run. The data format and complexity makes the scientific interpretation challenging and stressful. We need a better way of summarizing and presenting the data for easier interpretation. Unfortunately, there are no tools available to comprehensively visualize the genotyping outputs. We are developing a unified software tool to provide run summary metrics, genotype matrix table, genotypes in TOP/BOTTOM format, and additional features to view and compare the genotype calls.

Preliminary toolkit consists of the following features:

  • Genotype Summary—A summary report of the sequencing run with the high-level metrics of the sample call rates
  • GBSmatrix—Actual genotype alleles are displayed in a sample-by-marker matrix of all the samples from a single sequencing run.
  • GenotypeTB (TOP/BOTTOM)—By default, AgriSeq reports genotype calls based on the positive strand alleles. To compare different genotyping technologies and calculate concordances, genotype calls are converted and displayed in TOP/BOTTOM format.

The plugin will help researchers to interpret and troubleshoot the genotyping results more efficiently, and help facilitate the downstream analysis (e.g., GWAS, concordance studies with orthogonal technologies) by providing compatible data. The data visualization toolkit will bedistributed as an Ion Torrent Software Suite Plug-In.

For Research Use Only. Not for use in diagnostic procedures.

Haktan Suren, Krishna Reddy Gujjula, Prasad Siddavatam, Jason Wall, Rick Conrad Jeanette Schmidt

AgriSeq targeted genotyping-by-sequencing (GBS) is being used as a high-throughput, customizable and cost-effective genotyping solution in animal and plant breeding studies, parentage testing and genetic purity. One of the powers of this technology is its capability to support different types of markers, including single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphisms (MNPs), insertions and deletions (indels), and other structural variants (e.g., inversions, duplications). Long indels, which are longer than 100 bp, require a different strategy during the panel design and genotype calling. We employed a three-amplicon strategy to facilitate genotype calls from both the alleles and developed a new pipeline to get present/absent calls. We successfully developed a custom canine SNP genotyping panel with 16 long indels and evaluated the performance with known true genotype samples.

The robustness of this technology has been demonstrated across 384 samples using 16 canine long indel markers whose length ranges from 62 bp to 6 Mbp. Overall, 90% call rate across samples and 100% concordance calls with true genotypes were observed. We found that primer design and downstream analysis were not impacted by the indel size.

High call rate across multiple samples with varying indel size indicates the reproducibility and flexibility of the method. AgriSeq targeted GBS offers customers an end-to-end solution for genotyping diverse marker types simultaneously using the same workflow.

For Research Use Only. Not for use in diagnostic procedures.

Krishna Reddy Gujjula, Livio Baselgia, Jason Wall, Haktan Suren, Prasad Siddavatam, Rick Conrad and Jeanette Schmidt

Amplicon-based targeted genotyping-by-sequencing (GBS) has helped to enable scientists to screen for known variants in a high-throughput and cost-effective way. GBS with AgriSeq provides greater flexibility to study a set of known markers (SNPs, indels and structural variants) that can be multiplexed in a single genotyping panel. We evaluated the performance of AgriSeq technology for genotype call rate, concordance between replicates, and ability to identify novel variants within the targeted regions. We evaluated seven GBS panels ranging from 377 to 5,736 markers that represent both animal and plant kingdoms.

The average genotyping marker call rate across all panels was approximately 95% and the average genotyping marker concordance between replicates ranges from 96% to 99%. In addition to the genotype calls on the investigating markers, AgriSeq technology reports several novel (de novo) genotypes from the targeted amplicon regions. The average novel genotype concordance between replicate samples was 80 to 97%. Moreover, the proportion of novel calls as variants in publicly available databases (NCBI and Ensembl) was statistically significant (p-value << 0.01) than that could be explained by random error. This shows the biological relevance of the novel calls.

The high concordant calls for hotspots and novel genotypes indicate that the AgriSeq results are highly reproducible while maintaining high call rates. The high reproducibility of results shows the robustness of AgriSeq technology in spite of several sources of variations such as sample heterogeneity, library prep, sequencing runs and the human error. The ability to design panels ranging from a few hundred to several thousand genotypes for both animals and plants shows the versatility of the AgriSeq technology.

For Research Use Only. Not for use in diagnostic procedures.

Victor Missirian, John D. Curry and Ali Pirani

The Applied Biosystems Eureka genotyping solution is a low cost, high-throughput targeted genotyping-by-sequencing platform that supports the detection of tens to thousands of genetic markers (SNPs and insertions/deletions). It has been successfully used for a variety of applications (parentage, sex validation, genomic evaluation, carrier diseases) both in crops and animals.

After sample processing, Eureka next-generation sequencing (NGS) read counts are obtained for each sample, at each genetic marker. These counts are appropriately scaled, normalized, and transformed, before calling genotypes in a cluster-based Bayesian framework (BRLMM-P). The process can rapidly and reliably enable high-quality genotyping results across a wide range of panel sizes.

We demonstrate on four Eureka panels of varying size—two in the low range (tens to 500 markers) and two in the high range (500 to 3,000 markers)—that as panel size increases, we continue to obtain high sample pass rate, call rate, and concordance to known genotypes.

For Research Use Only. Not for use in diagnostic procedures.

Angela Burrell, Prasad Siddavatam, Michelle Swimley, Chris Willis, Haktan Suren, Krishna Gujjula and Rick Conrad

Parentage testing and genomics-assisted breeding are critical aspects of successful veterinary management. Due to its highly accurate and reproducible results, targeted GBS is becoming an increasingly favored technology for SNP genotyping. With the utilization of next-generation sequencing, labs can test hundreds of samples across thousands of SNPs simultaneously in a simple high-throughput workflow starting from either extracted nucleic acid or crude lysis samples. 

We developed a targeted sequencing panel, one for the combined detection of feline genetic defect/trait detection and parentage verification. Utilizing the AgriSeq HTS Library Kit, a high-throughput targeted amplification and re-sequencing workflow, the panel’s performance was tested on >96 diverse DNA samples. Libraries were sequenced on the Ion S5 platform using an Ion 540 chip with genotyping calling generated using the Torrent Variant Caller (TVC) plugin.

The mean genotype call rate of markers across the samples was >95%. Concordance across replicate library preparations and independent sequencing runs was >99% for both panels. Panel results also were compared with genotyping results from qPCR, and/or CE sequencing for orthogonal confirmation of accuracy, and the genotype calls were 100% concordant with the AgriSeq workflows. 

The data demonstrates the utility of the AgriSeq targeted GBS approach for feline SNP genotyping applications.

For Research Use Only. Not for use in diagnostic procedures.