International Society for Animal Genetics (ISAG) Conference

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.

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.

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%.