International Society for Animal Genetics (ISAG) Conference 2019

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 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 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 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.

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 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 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 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.

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 100bp, 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 62bp to 6Mbp. Overall, 90% call rate across samples and 100% concordance calls with true genotypes were observed. We found that primer design and down-stream 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 end to end solution for genotyping diverse marker types simultaneously using same workflow.

Microsatellites, also referred to as short tandem repeats (STRs), are the gold standard for parentage testing and identification including forensic analysis due to their Mendelian inheritance, and associated mutational diversity. Recently, the International Society of Animal Genetics (ISAG) added 3 new markers to the original 19-marker recommended panel for canine parentage determination, for a total of 22 loci. The Canine ISAG STR Parentage Kit (2014), from the AgriBusiness group at Thermo Fisher Scientific, is an optimized reagent kit for the analysis of the 22 STR loci recommended by ISAG in 2014.

The Canine ISAG STR Parentage Kit (2014) includes primer mix, amplification master mix, and canine gDNA control. The kit allows for the simultaneous amplification of the 22 ISAG recommended STR loci in a single multiplex amplification reaction. Buccal swab and oral fluid samples were prepared using various DNA extraction kits following the protocols recommended by the manufacturer. Amplicons are sized using capillary electrophoresis on Applied Biosystems genetic analyzers and GeneMapper software is used to determine each animal’s unique genetic profile.

The performance of the Canine ISAG STR Parentage Kit (2014) has been demonstrated across more than 42 canine breeds, multiple different sample preparation methods, and across different genetic analyzer capillary electrophoresis instruments. The complete workflow takes less than one day from sample to results, and several samples can be analyzed together, depending on the genetic analyzer instrument capabilities.

The Canine ISAG STR Kit (2014) enables customers to comply with ISAG recommendations with efficient out-of-the box optimized multiplex amplification reaction and fragment analysis while reducing risk of supporting complex homebrew panels.

Traditionally, high-throughput genotyping has been carried out by array based technologies. AgriSeq GBS (Genotyping-By-Sequencing) with Ion Torrent sequencing technology offers a faster, flexible, multiplexing, customizable, cost-effective alternative solution to study fifty to five thousand markers. AgriSeq GBS also provides capability to multiplex up to 1536 samples into 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 intrepretation. 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:

1. Genotype Summary—a summary report of the sequencing run with the high-level metrics of the sample call rates
2. GBSmatrix—actual genotype alleles are displayed in a sample-by-marker matrix of all the samples from a single sequencing run
3. 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 facilitate the down-stream analysis (e.g., GWAS, concordance studies with orthogonal technologies) by providing compatible data. The data visualization toolkit will be distributed as an Ion Torrent Software Suite Plug-In.