International Plant & Animal Genome XXV Conference (PAG)

View our PAG 2017 workshop presentations and scientific posters

A number of inescapable global dynamics are changing and will continue to change the agriculture and food ecosystem. The simplicity, accessibility, and ease of use of molecular technologies and NGS are reshaping the animal and plant breeding sciences. Our products and services offer high-quality, innovative life science solutions for every plant and animal research or breeding lab. Please view our PAG 2017 presentations to learn how Ion AmpliSeq genotyping by sequencing (GBS) and PCR solutions help address agricultural challenges.


Welcome & introduction

Ravi Ramadhar

Director of Agrigenomics

Thermo Fisher Scientific    


Thermo Fisher Scientific and Informa agribusiness insights and outlook: Challenges and opportunities in agribusiness—an economic perspective on global and technological factors transforming and impacting agribusiness

Tom Scott

Chief Executive Officer

Informa Agribusiness Intelligence IEG    

The world’s agriculture market is dynamic and subject to constant change. Investments in agricultural technologies and innovation are driven by the underlying economics of commodities and the markets they support. This session will provide both short- and long-term perspectives on the underlying agriculture market drivers and impact of the mega trends, trade, industry consolidation, and other economic drivers in agriculture—and the impact of these factors on innovation and technology in agriculture.

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Yield testing in the lab

Tom Osborn

Director of Molecular Breeding Technology

Monsanto

Genotyping at Monsanto has transformed through a series of technology innovations to the current state of automated workflows that deliver high-volume genetic data for key decisions in product advancement. Some recent innovations utilizing genome-wide selection and data from genotyping by sequencing have led to our ability to conduct yield testing in the lab. The success of this new workflow is dependent on collaborations with internal and external technology partners to deliver scalable, low-cost, and rapid lab processes.    

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Enabling more accurate animal parentage and genetic testing through targeted GBS and CE approaches

Wim van Haeringen, PhD

Chief Executive Officer

VHLGenetics

As a pioneer with a successful history in applying molecular technologies to animal genotyping, VHLGenetics is again leading the way with the application of genotyping by sequencing (GBS). VHL will present the output of a collaborative effort with Thermo Fisher Scientific on feline, canine, and bovine GBS panels for detecting genetic variants and parentage testing.

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A five-species 50K Axiom SNP microarray allows high-quality genotyping of coffee, cashew, cassava, brazilian pine, and eucalyptus

Dr. Dario Grattapaglia

Senior Research Scientist and Project Leader

Brazilian Agricultural Research Corporation (Embrapa)    

The identification of new sources of genetic variation is a key component of any long-term breeding strategy to increase the productivity, resilience, and sustainability of crop varieties. Over six million crop accessions are currently being conserved worldwide in still largely untapped genebanks. High-throughput, low-cost, and robust SNP genotyping technologies are necessary to unlock this wealth of diversity and release the data in an organized fashion to allow user-friendly adoption by germplasm curators and breeders.

Although more targeted and robust GBS methods have been developed, fixed content SNP arrays indisputably provide the gold standard for user-friendly, interchangeable, and fast-turnaround SNP data. To defray the upfront cost of developing an SNP microarray, we have built and validated a multispecies 50K Axiom SNP microarray. The array real estate was shared among five species according to the marker density needed, and the SNPs were selected for genetic applications including high-density linkage mapping, genomic selection, pedigree reconstruction, and germplasm characterization. From a total of 1,918 samples distributed across the five species, 1,817 (94.7%) were successfully genotyped, showing that the Axiom technology is robust across variable DNA sources. The SNP content was distributed as follows: 26,546 SNPs for coffee, 16,504 for cashew, 3,417 for cassava, 3,400 for araucaria (Brazilian pine), and 2,000 for eucalyptus. All SNPs were newly developed from whole-genome (cashew, coffee, cassava) or RAD and RNAseq (araucaria) data while previously validated SNPs were used for eucalyptus as control. SNP conversion rates were 84% for cashew, 94% for cassava, 93% for coffee, and a lower 74% for araucaria, likely due to the very complex and huge 23Gbp genome and limited RAD and RNAseq data used for SNP discovery. For eucalyptus, 92% of the SNPs were successfully converted from the Illumina Infinium platform. This microarray has enabled a number of powerful genetic studies in breeding and conservation, and represents the successful demonstration needed to build a larger array to accommodate SNPs for twelve species toward very large scale genebank genotyping at Embrapa.    

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Applications of Axiom microbiome arrays in food safety

Emily Rose

Sequencing Lab Supervisor

GeneSeek-Neogen    

Bacterial classifications and phylogenetic studies have been largely improved by the targeted amplicon sequencing of the hypervariable regions within the 16s rRNA gene, which is represented in all bacteria. However, limiting the classification investigation to solely 16s sequencing often still leads to difficulty in discerning closely-related organisms down to the species level. To aid in the pursuit of accurately identifying bacterial species and serotypes (strains), Affymetrix (a part of Thermo Fisher Scientific) has recently created the Axiom Microbiome Array as an effective assay to distinguish not only bacteria, but thousands of microorganisms at the species and even serotype level. The Axiom Microbiome Array is a highly developed microarray technology which incorporates >130,000 unique probes in order to detect over 11,000 individual microorganisms—including bacterial, protozoal, viral, fungal, and archaeal species—whereas 16s sequencing is strictly limited to the detection of bacterial organisms within a given sample. While 16s sequencing still holds some advantages in the field of bacterial classification, Neogen Corporation, a provider of food and animal safety solutions, recognizes the many unique applications of the Axiom Microbiome Array. This extensive-coverage array is a cost-efficient microbial analysis that will allow Neogen to provide quality solutions for agricultural industries such as animal health, as well as soil and feed quality. The application of the Axiom Microbiome Array technology will also have a significant impact on the crucial business of food safety and the importance of accurate microbial detection in edible products.

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Scientific posters

Angela Burrell, Michelle Swimley, Chris Willis, Adam Allred, Prasad Siddavatam, and Daniel Kephart

Genotyping by sequencing (GBS) has become an invaluable tool in animal and plant genomics to identify disease susceptibility and favorable traits, or even to determine parentage. GBS is typically performed on next-generation sequencing (NGS) platforms using either restriction enzyme–mediated techniques or through amplicon resequencing. Applied Biosystems AgriSeq library prep is a powerful, flexible, and customizable amplicon resequencing workflow which utilizes ultrahigh multiplex PCR for targeted sequencing of known SNPs, MNPs, and indels.

Library preparation is one of the most costly and time-consuming steps in the sequencing process. We have developed a 384-well library prep workflow for amplicon libraries that utilizes half the reaction volume of traditional 96-well workflows. Decreasing the reaction volume and increasing library throughput significantly decrease workflow costs. We have incorporated a pooling step into the workflow that allows the bead-based clean-up from up to four 384-well plates to be completed on a single 96-well plate, resulting in significant time savings. Library processing time was reduced by >1 hour with >50% reagent usage.

Performance of the high-throughput AgriSeq library prep workflow was validated with multiple Ion AmpliSeq panels. 384 barcoded samples were processed with the high-throughput AgriSeq library prep workflow and a standard volume 96-well workflow, and sequenced on the Ion S5 XL System. Equivalent performance was achieved between the two workflows including genotyping call rate, mean coverage depth, and coverage uniformity. The high-throughput AgriSeq library prep workflow provides a fast and economical alternative to larger volume, lower throughput library prep methods without sacrificing performance.    

Prasad Siddavatam, Angela Burrell, Adam Allred

Historically, microsatellites have been the most popular genetic feature for distinguishing cattle breeds for the purpose of determining parentage. More recently, SNP genotyping has emerged as a desirable alternative to microsatellite typing. SNPs offer several advantages over microsatellites. Perhaps the most important advantage is that there is less ambiguity in distinguishing SNP alleles in order to confidently provide a genotype call.

Ion AmpliSeq sequencing technology can be used for targeted amplification and resequencing of thousands of SNP targets in a single reaction. The Ion 540 chip allows hundreds of samples to be simultaneously genotyped at thousands of loci. Ligating a unique barcode to each sample allows samples to be sequenced together in a single run on the Ion S5 sequencing system.

We have developed a targeted sequencing panel based on 200 bovine SNP markers selected by the International Society of Animal Genetics (ISAG) for the purpose of determining parentage. We tested this panel on 96 bovine samples obtained from the USDA, representing 19 different breeds. Each sample was tested in duplicate, such that 192 libraries were pooled onto a single Ion 540 chip for sequencing. Variant calling was performed using the Torrent Variant Caller (TVC) plugin as part of the Torrent Suite software package. Mean call rate for this dataset was 98.5%, indicating that the vast majority of SNPs yielded data of sufficient quality to make a genotype call.

Ali Pirani, Vineet Joshi, Victor Missirian, and Teresa Webster

The 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 crop and animal agrigenomics testing.

The Eureka NGS read counts are appropriately scaled, normalized, and transformed to process genotyping in a cluster-based framework. The BRLMM-P two-dimensional clustering algorithm, which was originally developed for genotyping single-nucleotide polymorphisms (SNPs) and insertion/deletions (indels) on mid- to high-plex microarrays, has been extended for application on NGS reads. The algorithm has successfully been implemented across panels designed to study many organisms, and it incorporates methods for accurately genotyping samples originating from normal and inbred populations.

The Bayesian step in BRLMM-P gives it the flexibility to automatically adapt to cluster patterns exhibited by diploid, and various levels of allopolyploidy. It adapts pre-positioned genotype cluster locations called “priors” to the sample data and computes three posterior cluster locations. Genotype cluster locations are defined by two-dimensional means and variances for AA, AB, and BB genotype cluster distributions. Priors can be generic, meaning the same pre-positioned location is provided for every SNP; or SNP-specific, meaning different pre-positioned locations are provided on a SNP-by-SNP basis.

Eureka NGS read count ratios, similar to microarray intensities, have been shown to exhibit consistency across batches, resulting in repeatable spatial properties of genotype clusters. This allows SNP-specific priors, developed under controlled environments, to generalize. The process of prior development and genotype performance improvement is discussed.    

Michael Karberg, Prasad Siddavatam, Adam Allred, Daniel Kephart, Maarten de Groot, and Dr. Wim van Haeringen

DNA-based tests are commonly used to determine animal pedigrees. 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 successfully used for animal genetic identification, traceability, and paternity, although in recent years, single nucleotide polymorphisms (SNPs) have been increasingly used for this purpose. Genotyping by sequencing (GBS) using AgriSeq target enrichment technology in combination with the Ion S5 sequencing system allows one to simultaneously and accurately interrogate a large number of genetic loci (SNPs/indels) to identify the heritage of an animal, as well as screen for known genetic defects. In addition to the ability to screen for both animal heritage and disease-related genetic defects, advantages of this GBS approach include increased accuracy and improved assay flexibility (i.e., the ability to easily add or remove genetic loci being interrogated) which is desirable to both animal owners and breeders.

Here we describe the development of Ion AmpliSeq panels targeting canine and feline SNP markers for the purpose of determining parentage and genetic health. We tested these panels on samples derived from buccal swabs by sequencing them as a multiplexed (barcoded) pool on an Ion 540 chip. Variant calling was performed using the Torrent Variant Caller (TVC) plugin as part of the Torrent Suite software package. The mean call rate for this dataset indicated that the majority of SNPs were of sufficient quality to make a genotype call.

John Curry, Vineet Joshi, Qian Wan, Heather Koshinsky

The Eureka Genotyping Solution is a novel technology that has utility in plant breeding and regulatory aspects. It allows for a two-day turnaround time, from sample to genotype calls for catalog or fully customizable panels of a few 100s up to 3,000 variants (substitutions or indels). The Eureka genotyping assay is a ligation-dependent polymerase chain reaction made ready for high throughput sequencing. It consists of probe triplets in solution, where for each variant there are two left-side probes (one for each allele) immediately followed by a third right-side probe. The probes are hybridized to target on the input DNA and a ligation step locks the correctly hybridized left and right probes. Interrogation site bar codes are contained within the ligation probes, and sample index bar codes are added during the postligation amplification step. Amplicons from all the samples are pooled and short-cycle sequence data is generated from the prepared libraries. The Eureka™ Analysis Suite software is used to tabulate the number of reads that contain each combination of sample, locus, and allele bar code (as appropriate). The genotype of each sample for each locus is inferred from statistical analysis of the tabulated reads. The Eureka Genotyping Solution is affordable, flexible, accurate, and robust. Results for Eureka Genotyping Panels developed for Hordeum vulgare, Glycine max, Zea mays and the genetically more complex Triticum aestivum will be discussed.

Heather Koshinsky, Ali Pirani, Victor Missirian, Vineet Joshi, Qian Wan, Mark Stapleton, John Curry

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 is 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 sequence for high-value individuals.

The Eureka™ Ovine Parentage Panel is a comprehensive parentage panel for sheep with 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 that has the potential to increase overall revenue.