Tradeshow
Jul 07, 2019 - Jul 12, 2019
University of Lleida, Lleida, Spain
ISAG 2019-Hero

Workshop

Generations and genetics: Advancements in genetic tools for animal genotyping

Genomic technology continues to evolve and significantly impact animal genotyping and parentage verification.  During the workshop, industry leaders will discuss the applications of advancements in next-generation sequencing, content curation, and software analysis tools applied to ovine genomics selection and trait verification, as well as equine parentage verification.  As a partner committed to advancing and serving animal genotyping, Thermo Fisher Scientific provides innovative tools, and flexible and economical solutions to laboratories and breeders to accelerate animal genotyping.

Monday, July 8, 13:30–14:15
Auditorium 2, University of Lleida

Brenda Mae Murdoch PhD

Cost effective and informative genotyping by sequencing using AgriSeq targeted sequencing for genotyping in the livestock industry

Brenda Mae Murdoch PhD
Assistant Professor, Animal and Veterinary Science, University of Idaho

Download presentation

Genomic tools have enabled the acquisition of information to aid and accelerate the rate of genetic improvement of agricultural livestock species. With price still a limiting factor to widespread utilization in the U.S. sheep industry, there is a need for a lower density panel that reports markers for Mendelian and other well-known traits relevant to sheep production. The objective of this project was to design a genetic marker panel using AgriSeq amplicon targeted Genotyping By Sequencing (GBS). A marker DNA panel was designed to provide genotypic data for known single gene causative mutations, pedigree assignment and markers associated with quantitative trait loci common to the Axiom Ovine Genotyping array (50K). These markers are evenly spaced throughout the genome and located in either a transcript or quantitative trait locus. The flanking sequences of each marker was mapped to sheep reference genomes Oar_v3.1 and Oar_rambouillet_v1.0 to ensure the unique mapping of sequencing reads for data analyses. Both targeted and novel mutations within the sequenced region were called using Ion Torrent Suite 5.10. After excluding under-performing samples and markers, targeted markers with 50X sequence coverage or greater and an overall average call rate of 98% were included in the data set. Data from this panel will be used to identify known causative mutations, improve pedigree accuracy for the sheep industry. The application of this cost-effective sheep genomics marker panel can be used to promote improved production, animal health and profitability of the U.S. sheep industry.

Paul Flynn  Head of R&D

Development of an AgriSeq targeted GBS panel for equine SNP parentage verification and sire/dam allocation

Paul Flynn
Head of R&D, Weatherbys Scientific

Download presentation

Transitioning from STR to SNP molecular markers to perform parentage verification is becoming an ever more discussed topic within the global Equine community. A key area that requires exploration is ensuring maintenance of test integrity when moving to SNP based parentage. Due to its highly accurate and reproducible results, targeted GBS is becoming an increasingly favoured technology for SNP genotyping. AgriSeq GBS with Ion Torrent NGS technology offers a fast, flexible, multiplexing, customizable, cost-effective solution to study hundreds of samples across fifty to five thousand markers within each genotyping run using either extracted nucleic acid or crude lysis.

We developed a targeted ~500 SNP Equine GBS panel consisting of ISAG parentage verification SNPs and an additional SNP panel for Sire/Dam allocation. The panel design utilised latest reference genome build - EquCab3.0, with corrections for markers mapping to previous reference genomes EquCab1 and EquCab2. Utilising AgriSeq HTS Library Kit, a high-throughput targeted amplification and re-sequencing workflow, the panel’s performance was tested on a diverse set of Equine Thoroughbred DNA Sire/Dam/Offspring Trios. Libraries were sequenced on Ion S5 using an Ion chip with genotype calls generated using Torrent Variant Caller (TVC) plugin. All Equine animals were also genotyped for 16 STR markers (inclusive of 12 ISAG STRs) which allowed for comparative assessment of parentage efficacy and Sire/Dam allocation accuracy between SNP and STR markers. Furthermore, previously demonstrated flexibility of AgriSeq GBS panel design also facilitates considerations for additional SNPs for diagnostic traits and STR imputations to future panel versions.

Results of the panel performance including mean genotype call rate of markers across samples, concordance across replicate library preparations and independent sequencing runs, parentage efficacy and Sire/Dam allocation accuracy results are reported. The data demonstrates Equine SNP parentage in practice and the utility of the AgriSeq targeted GBS as a platform to support Equine SNP parentage verification genotyping needs.

Scientific presentations

Development of targeted GBS panels for breeding and parentage applications in dogs

Angela Burrell, Prasad Siddavatam, Michelle Swimley, Chris Willis, Haktan Suren, Krishna Gujjula, and Rick Conrad
Session: Applied Genetics of Companion Animals
Location: Room 5
Date/Time: Tuesday, July 9, 10:15 am
Duration: 15 minutes

Download poster

Download presentation

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.

Development of targeted GBS panels for breeding and parentage applications in cats

Angela Burrell, Prasad Siddavatam, Michelle Swimley, Chris Willis, Haktan Suren, Krishna Gujjula, and Rick Conrad
Session: Applied Genetics of Companion Animals
Location: Room 5
Date/Time: Tuesday, July 9, 10:00 am
Duration: 15 minutes

Download poster

Download presentation

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.

End-to-end targeted GBS long INDELs Solution

Haktan Suren, Krishna Reddy Gujjula, Prasad Siddavatam, Jason Wall, Rick Conrad, Jeanette SchmidtSession: Applied Genetics of Companion Animals
Session: Applied Genetics of Companion Animals
Location: Room 5
Date/Time: Tuesday, July 9, 11:15 am
Duration: 15 minutes

Download poster

Download presentation

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.

Development of targeted GBS panels for breeding and parentage applications in cattle and swine

Angela Burrell, Prasad Siddavatam, Michelle Swimley, Chris Willis, Maarten de Groot, Ryan Ferretti, and Rick Conrad

Session: Cattle Molecular Markers and Parentage Testing
Location: Room 3
Date/Time: Tuesday, July 9, 4:30 pm
Duration: 20 minutes

Download poster

Download presentation

Parentage testing and genomics-assisted breeding are critical aspects of successful herd 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 targeted sequencing panels for both cattle parentage, based on 200 SNP markers selected by the International Society of Animal Genetics (ISAG), and swine breeding using a 1500 SNP imputation panel.  Utilizing the AgriSeq HTS Library Kit, a high-throughput targeted amplification and re-sequencing workflow, each panel’s performance was tested on >96 diverse cattle and swine 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 >98% for the cattle panel and >96% for the swine panel.  Concordance across replicate library preparations and independent sequencing runs was >99.9% for both panels.  Panel results were compared with results from a DNA array and the genotype call concordance was >99% with the AgriSeq workflows.  The cattle panel was also used on field samples by a Netherland service lab to successfully determine the parentage relationships of 45 calves with 48 potential mother cows.

The data demonstrates the utility of the AgriSeq targeted GBS approach for cattle and swine SNP genotyping applications.

Scientific posters

Optimized fragment analysis kit to determine canine parentage with ISAG-recommended STR markers

Denisse Meza, Robert Tebbs, Rick Conrad

Download poster

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.

Genotyping data visualization toolkit for targeted AgriSeq GBS

Prasad Siddavatam, Haktan Suren, Krishna Reddy Gujjula and Jeanette Schmidt

Download poster

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.

AgriSeq targeted GBS is a customizable high-throughput genotyping technology that permits fast, easy, and inexpensive alteration of marker content

Claudio Carrasco, Krishna Reddy Gujjula, Haktan Suren, Prasad Siddavatam, and Christopher C. Adams

Download poster

Attractive and valuable high-throughput genotyping solutions for parentage and breeding applications require the ability to simultaneously interrogate hundreds to thousands of genetic loci both easily and economically. One disadvantage of many high-throughput genotyping technologies is the lengthy lead times and considerable cost associated with changing the genomic marker content (targeted loci) in any particular assay. The Applied Biosystems AgriSeq targeted genotyping-by-sequencing (GBS) solution for plant and animal genotyping does not suffer from this problem because the technology relies on a pool of PCR oligonucleotides that can be quickly, easily and inexpensively changed to accommodate always improving knowledge of genomic function. If and when the need arises to alter the content of a marker panel all that is required is the design and synthesis of additional PCR primers which are then simply spiked into existing assay pools. In addition, AgriSeq genotyping panels can be ordered in plate format in which primer pairs for marker-containing amplicons are individually aliquoted enabling the user to drop unneeded amplicons or re-formulate primer pools (panels) in any combination desired. Furthermore, individual panels targeting specific species can mixed together, creating a multi-species panel, while still enabling species-specific genotyping. For example, a mixture of three mid-density panels for multiple species not only allowed for accurate species-specific genotyping, but also enabled the accurate assignment of species to unknown gDNA samples being tested. Moreover, the flexibility of the workflow allows users to mix AgriSeq libraries from different species on a single chip for sequencing, which improves the overall economics. This unparalleled flexibility in a highly multiplexed genotyping platform provides users unlimited avenues for customizing their genotyping workflows.