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AgriSeq Targeted Genotyping By Sequencing
Innovative solutions that help accelerate plant and animal breeding programs
Molecular breeding can significantly reduce the cost and time required to deliver improved plant and animal species for agricultural use. Advancements in genomic technologies are accelerating these breeding programs by enabling higher-throughput genotyping across large populations than ever before. Applied Biosystems AgriSeq targeted genotyping by sequencing (GBS) solutions are our latest innovation that helps our customers economically deliver high-throughput plant and animal genotypes.
Up to 2.6M genotypes per day for pennies or less per genotype
The AgriSeq targeted GBS solution utilizes a highly efficient multiplexed PCR chemistry where hundreds to thousands of markers can be targeted and uniformly amplified in a single reaction. The amplicon libraries can then be barcoded and pooled for simultaneous sequencing of hundreds of samples on the Ion Torrent suite of next-generation sequencing (NGS) instruments. At a cost of pennies or less per genotype, AgriSeq targeted GBS is capable of generating up to 2.6 million genotypes per day from high-quality NGS data. Whether you’re looking to achieve more accurate marker-assisted breeding selection, or to improve your livestock parentage discrimination and trait monitoring, our solutions can help advance your plant and animal breeding programs.
Scalable genotyping
AgriSeq genotyping is performed on the Ion Torrent NGS platform and is validated for 100–5,000 markers per sample. Using the Ion 540 chip, AgriSeq genotyping can produce up to 1.3 million unique genotype results per chip at an average read depth of 100x to achieve maximal genotype call rates. Combinations of different panels and species can also be processed on the same run.
Reproducible results
Deliver high marker call rates with greater reproducibility across diverse sample sets to help ensure accurate selection. Marker call rates can exceed 96% with >99% inter- and& intra-run reproducibility, and AgriSeq targeted GBS data maintains a high concordance (>99%) when compared to the orthogonal genotyping technologies such as DNA microarrays.
Fast, simple workflow
Includes library generation, automated template prep, sequencing, and data analysis, which can be completed in ~2-3 days with <4 hours of hands-on time, enabling a single operator to process up to four 96- or 384-well plates per day. The Ion Chef System automates template preparation and chip loading, requiring only 15 min of hands-on time, followed by an overnight run.
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Easy data summarization and visualization
Ion Torrent™ AgriSum Tool Kit (AST) plug-in aggregates data from the coverageAnalysis and variantCaller plugins for easy review of data. AST displays overall sequencing run summary metrics for easy review, enabling quality control or troubleshooting, if needed. Additionally, AST converts TVC data into genotype Martix, TOP, and TOP/BOTTOM formats for further analysis applications.
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AgriSeq companion animal and livestock genotyping solutions
Key features of AgriSeq ready-to-use panels include:
- Reproducible genotyping of ISAG-recommended markers for all species with high call rates
- Simple, high-throughput workflow
- Customizable approach to combine with compatible parentage, traits or disorders, or additional markers of interest
AgriSeq Canine Traits and Disorders Panel
The AgriSeq Canine Traits and Disorders Panel contains 154 markers targeting clinically important genetic traits and disorders in canines. The panel includes 97 SNPs and complex markers such as 6 MNPs, 13 insertions, and 38 deletions. Most of these markers are common to all breeds, while some are breed specific for certain traits and conditions. The performance of the panel was verified using orthogonal testing, robustness testing, and field testing with diverse DNA samples from canine oral swabs. The panel enables multiplexed simultaneous genotyping of 154 markers to deliver reliable genotyping results for 136 genetic disorders and 18 traits using the AgriSeq targeted GBS workflow. The CanFam 3.1 reference genome is used for the analysis.
AgriSeq Canine SNP Parentage and Identification Panel
Contains 379 SNPs and 2 deletion markers designed to deliver reliable genotyping results for parentage The AgriSeq Canine SNP Parentage and Identification Panel contains 379 SNPs and 2 deletion markers designed to deliver reliable genotyping results for parentage determination and identity verification of canines by targeted GBS. The panel enables multiplexed simultaneous genotyping using the AgriSeq targeted GBS workflow. The performance of the panel was verified using orthogonal testing, robustness testing, and field testing with diverse DNA samples from canine oral swabs. The panel was also qualified using the 2019 ISAG/ICAR 2nd SNP Typing Canine Comparison samples. The CanFam 3.1 reference genome is used for the analysis.
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Figure 1. Performance of AgriSeq canine panels. Call rates were calculated for both panels after testing >180 diverse DNA field samples from canine oral swabs with the AgriSeq workflow. The mean call rate for the AgriSeq Canine Traits and Disorders Panel was 99.1% (A), and for the AgriSeq Canine SNP Parentage and Identification Panel was 99.2% (B), demonstrating the high performance obtained from customer samples.
AgriSeq Feline Parentage and ID Plus Traits and Disorders Panel
The AgriSeq Feline Parentage and Identification Plus Traits and Disorders (PITD) Panel is a combined panel for determining parentage and identification (111 SNP markers) plus targeting 64 clinically important traits and disorders (43 SNP, 1 MNP, 4 insertion, and 16 deletion markers) in felines by targeted GBS. The performance of the panel was verified using orthogonal testing, robustness testing, and field testing with diverse DNA samples from feline oral swabs (Figure 4). The 111 SNP parentage and ID markers were also qualified using the 2019 ISAG/ICAR 2nd SNP Typing Feline Comparison samples. The comprehensive AgriSeq Feline PITD Panel is designed to deliver reliable genotyping results for parentage determination, including identity verification and prediction of 42 genetic disorders and 22 traits that are clinically important. When desired, additional custom markers referencing the Felis_catus_8.0 can be added if compatible.
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Figure 2. Performance of AgriSeq Feline panel. A total of 258 feline oral swab DNA samples were tested either in duplicate or in quadruplicate with the AgriSeq workflow using the AgriSeq Feline PITD kit. The call rate, the number of markers generating a genotype call for each sample, was calculated for all samples with at least 100x mean read depth, the minimum recommended. The mean call rate was 99.3% and all samples had call rates >92.5%, demonstrating the high performance obtained from field samples.
Bovine ISAG SNP Parentage Panel (2013)
The Applied Biosystems Bovine ISAG SNP Parentage Panel (2013) contains primers targeting the 200 ISAG-recommended SNPs available for simple multiplexed analysis using the AgriSeq targeted GBS workflow. The ISAG has standardized a set of 100 core SNPs and 100 additional SNPs with sufficient minor allele frequency (MAF) and genomic spacing for accurate bovine parentage determination across a wide range of breeds (Figure 5). You can download SNP information from the ISAG website [3]. Qualified using the 2015 ISAG/ICAR 3rd SNP Typing Bovine Comparison samples, our comprehensive Bovine ISAG Parentage Panel is designed to deliver reliable genotyping results for animal parentage testing. The Bos taurus reference genome is needed for analysis of the Bovine ISAG SNP Parentage Panel and can be obtained by contacting our technical support specialists.
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Figure 3. Performance of AgriSeq Bovine panel. Evaluation of 96 diverse bovine gDNA samples from the United States Department of Agriculture (USDA), representing 19 different breeds with the Bovine ISAG SNP Parentage Panel (2013), with 4 replicates each. All 384 individually barcoded libraries were pooled onto a single Ion 540™ Chip and sequenced on the Ion S5™ XL System. All but one of the samples tested gave a mean sample call rate of >95%. The mean call rate across all samples was 98.5%.
Custom panel designs, validation and pilot projects
Agricultural genomics can be challenging for a number of reasons, including sequence complexity, high genetic diversity, and limited sequence availability. We offer end-to-end project management services through panel design, wet-lab validation, and data analysis to help ensure delivery of high-performance panels for your project. These low cost, proof of concept pilot projects are a great way to confirm the value of AgriSeq targeted GBS for your research.
IonCode Barcode Adapters
IonCode Barcode Adapters are a family of products that provides 1536 unique barcode adapters designed for optimal performance with Ion Torrent semiconductor sequencers. When used in combination with the AgriSeq HTS Library, users can pool up to 1536 amplicon libraries and then conduct multiplexed sequencing analysis, which helps increase lab efficiency and reduce sequencing costs per sample.
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Resources
Videos
Tom Osborn, Monsanto's Director of Molecular Breeding Technology, explains how genotyping has been transformed through a series of technology innovations to the current state of automated workflows.
For Research Use Only. Not for use in diagnostic procedures.