International Plant & Animal Genome XXV Conference (PAG)

It was nice to see you at the ISAG 2017 conference

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Our Workshop: Next-Generation Solutions for Animal Breeding and Parentage

Advances in genotyping and genomic technologies such as next-generation sequencing and microarray technologies are making it easier and more affordable than ever to genotype large populations of animals. Genomic evaluations provide insight into parentage, pedigrees, and other genetic effects that have both economic and animal health implications. Sequencing-based technologies like AgriSeq targeted genotyping by sequencing (GBS), and a microarray system such as Axiom microarrays, allow up to hundreds of thousands of markers to be interrogated simultaneously, improving selection accuracy and trait monitoring in animals.  

Please download our ISAG 2017 workshop presentations to learn how two genotyping labs have implemented AgriSeq targeted GBS and Axiom custom high-density microarrays in their genetic evaluation programs to improve lab efficiency, throughput, and cost per genotype for canine, feline, and ovine species.  

Ravi Ramadhar

Ravi Ramadhar

Product Manager Leader Agrigenomics

Thermo Fisher Scientific

Introduction to Genotyping Technologies

Donagh Berry

Quantitative Geneticist


Ovine Genotyping by Numbers

Sheep, because of their relatively low unit value and lack of widespread use of artificial insemination and single-sire mating, differ from many other farmed species in their requirements for low-cost genomic technologies. Therefore, because cost is crucial, sheep breeds must constantly be on the lookout and be able to easily change genotyping vendors, technologies, service providers and marker panels in search of lowest-cost genotypes. Two main genotyping vendors currently exist for large-scale (i.e., ≥10,000 SNPs) genotyping although new entrants to the market are emerging. The genotype concordance between 84 sheep genotyped for 51,121 SNPs on both Thermo Fisher Scientific and Illumina platforms was 0.971; this increased to 0.995 when restricted to only high-quality SNPs. Therefore, both platforms are very similar and can be used interchangeably in search of lower genotyping costs. The standard genotype panel used in genomic evaluations of sheep (and many other species) is around 50,000 SNPs. The International Sheep Genomics Consortium developed an Illumina panel with 15,000 SNPs which also included SNPs in genes with major effects, although not all of these genotyped with sufficient quality. An 11,136-SNP Applied Biosystems Axiom panel was recently developed, which included a sub-set of the 15,000 SNPs that were high-quality, segregating in several sheep breeds, displayed high quality metrics (as determined by both Thermo Fisher Scientific and Illumina statistics) and had good genotype concordance between the Axiom and Ilumina panel. The median minor allele frequency per breed (Texel, Suffolk, Vendeen, Charollais, Vendeen) varied from 0.29 to 0.33, with only up to 54 monomorphic SNPs found in any one breed. The accuracy of imputation (i.e., correlation between actual and imputed genotypes) of 376 of the youngest animals from a multi-breed reference population of 2676 sheep, using the Thermo Fisher Scientific and Illumina lower-density panels, to a 50,000-SNP panel, was 0.97881 and 0.97799, respectively. Thus, as well as including SNPs in genes conferring major effects (e.g., scrapie), the Axiom low density, low-cost sheep panel is suitable for use in imputation to higher density for accurate genomic evaluations. The correlation between allele counts from 162 SNPs on 89 animals genotyped using either the Applied Biosystems Eureka or Axiom genotyping platforms was 0.94; when 14 SNPs with poor concordance were not considered, the correlation strengthened to 0.97.

Maarten de Groot

Chief Financial Officer


Implementation of an AgriSeq Genotyping Workflow to Improve Efficiency of Canine and Feline Genetic Testing in a Service Lab

Genetic testing in companion animals has focused on parentage determination and monitoring of genetic defects common in different breeds. Traditional approaches have commonly utilized singleplex technologies on only the most common disorders as more comprehensive testing approaches using array technologies are cost-prohibitive. Recent advances in targeted next generation sequencing technologies are creating new opportunities for service labs to expand the breadth of variants evaluated in a single, low cost test. Here, we report our experience in implementing multiplexed panel approaches for canine and feline genetic defect and parentage testing. Using AgriSeq targeted GBS chemistry, we are able to interrogate 157 canine genetic defects and 200 parentage SNPs simultaneously in one test. Results obtained using the AgriSeq workflow have demonstrated high reproducibility as well as concordance with orthogonal genotyping methods. Finally, the flexibility of this approach allows different panels and different species to be analyzed together in the same sequencing run, greatly streamlining our lab efficiency and turn around time while increasing the depth and value of genetic information returned to our customers.

Posters & oral presentations

Next-Generation Targeted Sequencing Panel for Verification of Bovine Parentage

Angela Burrell, Prasad Siddavatam, Adam Allred, Michelle Swimley, Chris Willis, Ryan Ferretti, and Alex Raber. Thermo Fisher Scientific, Neogen GeneSeek Operations.

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Bovine parentage verification is a critical aspect of successful herd management.  Due to its highly accurate and reproducible results, SNP genotyping is becoming an increasingly favored tool for parentage verification. Using high-throughput next-generation sequencing platforms like the Ion S5™ sequencing system, labs can test hundreds of samples and thousands of SNPs simultaneously.

We developed a targeted sequencing panel based on 200 bovine SNP markers selected by the International Society for Animal Genetics (ISAG) for the purpose of verifying bovine parentage. Utilizing the AgriSeqTM sequencing workflow, a high-throughput targeted amplification and re-sequencing workflow, panel performance was tested on 115 diverse bovine DNA samples.  Samples included a panel of 96 samples obtained from the USDA (MARC Beef Cattle Diversity Panel v2.9) as well as samples contained within the 2015 ISAG/ICAR 3rd SNP Typing Bovine Comparison Test panel.  Samples originated from 20 different bovine breeds.  Libraries were prepared using the high-throughput AgriSeq workflow.  The resulting amplicons were ligated to unique barcodes and sequenced on a single run on the Ion S5™ sequencing system using an Ion 540™ chip.  With this system, up to 768 samples can be barcoded and run on a single sequencing run allowing for up to approximately 1500 samples to be tested a day (2 runs/day).  Data were analyzed using the Torrent Variant Caller (TVC) plugin as part of the Torrent Suite™ software package to determine the genotype call for each marker and sample. 

The mean call rate (the percentage of markers generating a genotyping call) was >98%, and genotyping call concordance between library prep replicates was >98%.  As a comparison, the USDA gDNA was also hybridized to six replicate arrays to generate a consensus array genotype call.  Genotype call concordance was >99% between the array and AgriSeq workflow calls.  Accurate parentage determinations were made for the samples within the 2015 ISAG/ICAR 3rd SNP Typing Bovine Comparison Test panel as compared to provided reference genotypes. The data demonstrate that the bovine parentage panel tested with the AgriSeq workflow provides accurate and reproducible results for SNP-based parentage verification.

High Cross-Platform Genotyping Concordance of Axiom High-Density Microarrays and Eureka Low-Density Targeted NGS Assays

Mohini A Patil, Ali Pirani. Thermo Fisher Scientific

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Microarrays ranging from mid- to high-plex have been developed on the Applied Biosystems Axiom Genotyping Solution to interrogate single-nucleotide polymorphisms (SNPs) and insertion/deletions (indels) for over 55 agrigenomic organisms. This technology has the flexibility to genotype populations exhibiting diploid to various levels of allopolyploid genetics. It also incorporates methods for accurately genotyping samples originating from normal and inbred populations. The high variant density possible on microarrays has the ability to facilitate multi-breed genomic selection, fine mapping of quantitative trait loci, and detection of copy number variation.

The Applied Biosystems Eureka Genotyping Solution is an affordable, low- to mid-plex, high-throughput genotyping assay that uses common next-generation sequencing (NGS) platforms for signal readout. It enables the detection of tens to thousands of genetic markers which are increasingly in demand for routine animal agrigenomics testing. This routine testing can include parentage and sex validation and genomic evaluation, after imputation.

High reliability and concordance across the Axiom and Eureka technologies is essential to allow seamless migration between the platforms. Thermo Fisher Scientific has accomplished high genotype concordance and high genotype call rates (low missing-data rates) by adapting the same genotype calling and SNP QC framework across both platforms.

The developed genotype-calling algorithm has been shown to work on both microarray intensity and counts of allele x locus barcodes of next-generation sequencing (NGS) reads. Various overlapping animal datasets have been evaluated across these microarray and targeted NGS technologies. A high level of genotype concordance is demonstrated, allowing for easy comparison across and migration between platforms.

AgriSeq Targeted Sequencing Panel for Determination of Canine Parentage and Genetic Health

M. Karberg, A. Burrell, P. Siddavatam, A. Allred, M. de Groot, W. van Haeringen. Thermo Fisher Scientific; VHLGenetics

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The objective of this presentation is to show the performance of Applied Biosystems AgriSeq primer panels for canine parentage and genetic health, and demonstrate how the panels can be combined, or otherwise modified, without detrimental effects to detection.

Ensuring an accurate pedigree is particularly important for purebreds, having both economic and animal health implications. Historically, microsatellites (short tandem repeats, or STRs) have been used for genetic identification, traceability, and paternity. In recent years, other DNA based tests such as single-nucleotide polymorphism (SNP) detection have become increasingly used for this purpose. High-throughput targeted amplification and re-sequencing, using the Applied Biosystems AgriSeq target enrichment technology and the Ion S5 sequencing system, allows for the simultaneous and accurate genotyping of a large number of SNPs to interrogate the heritage and genetic health of an animal. In addition, the AgriSeq approach is very flexible, allowing panels to be combined or modified easily, thereby helping both breeders and diagnostic laboratories stay relevant with evolving content needs.

Here, we describe the development of two AgriSeq panels targeting canine SNP markers: a parentage panel based on 200 ISAG canine targets, and a genetic health panel based on over 140 well-characterized genetic markers. To demonstrate the modularity of the AgriSeq approach, the performance of the genetic health and parentage panels was analyzed independently, and combined and analyzed simultaneously using 192 samples pooled on an Ion S5 540 chip. Variant calling was performed using the Torrent Variant Caller (TVC) plugin as part of the Ion Torrent Suite software package. The data show that the two panels work similarly, regardless of whether they are used separately, or combined together on one sequencing chip. The mean sample call rate was >95%, and the sample concordance between the separate and combined panels was >99.9%.

Accelerating Genetic Improvement in Sheep by Increased Pedigree Accuracy

Heather Koshinsky, Ali Pirani, Victor Missirian, Vineet Joshi, John Curry. Thermo Fisher Scientific.

Genetic management is a key driver for increased revenue in agrigenomics production systems. The amount of genetic information required for a decision depends on the application. At the low end are genotypes on a few hundred markers for a targeted SNP parentage and the associated reduction in pedigree errors and more efficient herd improvement. Correct parentage assignment increases the success of any breeding program by facilitating linkage of production performance to the correct families to improve estimates of breeding values. At the high end is full genome sequencing for high-value individuals.

The Eureka Ovine Parentage Panel is a comprehensive parentage panel for sheep and provides superior power to accurately verify parentage. It provides an affordable next-generation sequencing (NGS)–based panel for both parentage testing and traceability in diverse sheep breeds. The availability of over 3,000 barcodes enables processing of over 3,000 samples in a single sequencing run for fast turnaround time. Thus, this genotyping panel may be used as a tool in an ovine breeding and production system and has the potential to increase overall revenue.

New Output Formats for Axiom Genotyping Arrays

J. Foster, A. Davassi, A. Pirani, S. Kaushikkar, B. Wong, M. Patil, L. Jevons. Thermo Fisher Scientific

The high-throughput agricultural genotyping landscape encompasses a broad range of applications and technical platforms. One of the major challenges of adopting a new platform or performing meta-analyses is data format congruity. Bi-allelic genotypes are recorded in one of three ways: “AA”, “AB”, and “BB” call codes; “0”, “1”, and “2” numeric call codes; and base calls. 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 bi-allelic 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, we have developed the Affymetrix Axiom Long format Export tool (AxLE), a companion application to the Axiom Analysis Suite (AxAS). The tool formats Axiom genotype data from the native “Forward” format to the top (TOP) and bottom (BOT) designations based on the polymorphism itself, or the contextual surrounding sequence, and designates the A/B allele.

A clear requirement for the homogenization of allele designation is in the downstream application of genotyping data to genetic evaluation systems where mixing of the formats could be disastrous to the prediction of economically important traits. To support this specific use case in dairy cattle, we have developed the Affymetrix CDCB (Council on Dairy Cattle Breeding) export tool—a companion application to AxAS. Once a genotyping export has been formatted using AxLE, the CDCB export tool formats it and a sample sheet to enable direct upload to the Council on Dairy Cattle Breeding website. The tool is capable of consuming data from any Affymetrix bovine array and also custom bovine designs.