Plant & Animal Genome XXVI, PAG 2018

View our PAG 2018 workshop presentations and scientific posters

Diverse applications using smarter genomics

Genomic technology continues to evolve with advancements in next-generation sequencing, content curation, and software analysis tools. During the workshop, three customers and a Thermo Fisher Scientific employee discuss the applications. From high-throughput genotyping in cucumber breeding and use of next-generation sequencing to assess seed quality control in soybeans, to complex data analysis in polyploidy species and disease mapping in canines, Thermo Fisher Scientific provides innovative tools and solutions to accelerate plant and animal research.

Workshop

Advancing vegetable breeding with AgriSeq targeted genotyping by sequencing

Hans Peter Koelewijn

Scientist, genetics
BAYER Vegetable Seeds

Bayer Vegetable Seeds identified a need for a platform that would result in a greater throughput, a higher marker density (1000 to 10,000 SNPs) and a fast turnaround time to advance their breeding program. With a variety of vegetable crops and rapidly changing marker information, Bayer needed a flexible solution.

After a market evaluation of different technology options, Bayer elected to evaluate the AgriSeq targeted genotyping-by-sequencing solution. Its promise of high marker density, fast throughput, and flexibility was an attractive solution. Conducted as a collaborative pilot program, cucumber was chosen as the first crop to conduct a pilot study. The program process included panel design, optimization, and implementation.

This session will discuss the process and present results including marker call rate, workflow reproducibility, concordance with legacy genotyping results, and novel SNP discovery. The session will explore the power and promise of the AgriSeq solution, the impact of Agriseq technology on the cucumber breeding program, and implications for lab workflows and broader breeding programs.

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Determination of purity and quantification of varietal components through AgriSeq targeted GBS

Dr. Carlos Azambuja

Director
GENIA

The barley industry requires strict control of the varieties involved to maintain optimal malting procedure and limit batch variability.  We were asked to develop a method to enable the determination of varietal purity as well as to identify and quantify the components of varietal mixtures.

We speculated that, as each variety has its own SNP profile, we might be able to determine the percentage of each variety by determining the frequency of each allele in the pool sample. In order to determine the components of a varietal mixture, we developed an algorithm based on Bayesian method utilizing a database of the 43 varieties of barley most frequently used in Uruguay, Argentina, and Chile.

A set of 400 SNPs was selected from the iSelect 9k barley chip. The SNPs’s performance was tested in silico by the AgriSeq team; the final panel contained 369 SNPs. DNA was extracted from the flour of 10,000 seeds, and PCR and library constructions were processed according to AgriSeq protocols. Sequencing was performed on an Ion S5.

This method was validated by mixtures of known composition being able to determine varietal purity with an error rate lower than 1% (99% confidence) and 95–99% purity. The system can detect 0.8% of contaminant varieties using a coverage of 100X. The system was also validated using malted barley.

Using the same approach, we developed a soybean panel with 363 SNPs based on the BARCSOYSNP6K and tested it against 46 soybean varieties. The panel was able to discriminate all the varieties with an average of 80 differential SNPs between varieties, and a minimum of 4 differential SNPs in closely related varieties. The quantification capability of the AgriSeq system proved to be a reliable tool for varietal purity determination.

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Diverse applications of Applied Biosystems Axiom Genotyping Arrays in plant and animal breeding and research

Ali Pirani

Manager, Bioinformatics, Microarray
Thermo Fisher Scientific

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. An apt high-throughput genotyping platform can enable marker-assisted breeding, association mapping, and genomic selection. The Applied Biosystems Axiom genotyping platform provides extensive information on polymorphic variation across the genome of species of interest, and can be widely used to characterize genetic properties of diverse populations, fingerprinting, and QTLs. The platform has been successfully implemented in diploid breeding programs. However, while it is widely known that polyploidy is challenging for genotyping, we have successfully implemented the Axiom technology for complex species such as wheat, strawberry, sugarcane, and salmon.

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Investigating the genetics of mixed breed dogs with the new Axiom array

Elinor K. Karlsson, PhD

Assistant Professor
Program in Bioinformatics and Integrative Biology
University of Massachusetts Medical School;
Director of Vertebrate Genomics
Broad Institute of Harvard and MIT

Most dogs in the United States—and many dogs throughout the world—are mixed-breed dogs, yet past genetic studies in dogs have focused almost entirely on purebreds. In part, this reflected technological limitations, as the genotyping panels available were designed for purebred dog populations.

Here, we use a new, high-density Axiom array to genotype over 500 pet dogs that we collected through our Darwin’s Dogs citizen science project. We show that mutts are even more diverse than expected, with each dog a mosaic of many different breed ancestries. By capturing more of the genomic variation in each dog, the Axiom array enables powerful genome-wide association studies in populations that include mixed-breed and purebred dogs.

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Scientific presentations

Angela Burrell, Prasad Siddavatam, Adam Allred, Michelle Swimley, Chris Willis, Maarten de Groot, Ryan Ferretti, and Rick Conrad
Saturday, January  13, 2018

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 AgriSeqTM 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 Netherlands 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

R.C. Willis, A.Burrell, M. Swimley, P. Siddavatam, C. Buchanan-Wright, R. Conrad     

The utility of restriction-enzyme genotyping by sequencing (GBS) in production agriculture is challenged because of the technology’s limitations in SNP targeting and high rates of allele dropout between samples. In contrast, targeted Genotyping by Sequencing (GBS) can deliver consistent, high marker call rates for specified SNPs in a high-throughput, cost-effective manner. AgriSeq targeted GBS allows up to 5000 markers to be simultaneously screened across 100s of samples in a single Ion Torrent sequencing run. The robustness of this technology has been demonstrated across 19 agriculturally relevant species, with marker call rates between 88–98%, >99% reproducibility and >99% concordance with orthogonal genotyping technologies. Here, we report the expansion of the AgriSeq workflow to accommodate complex structural variants with in-dels ranging between 2–400 ntds.

Enhancements have also been made to the AgriSeq workflow to improve throughput. An additional 384 IonCode barcodes were developed to enable 768 sample multiplexing. Barcodes were validated over 3 different panels generating equivalent performance for ligation efficiency, uniformity, and mapped reads. In addition, AgriSeq automation workflows were developed on the Gilson PipetMax to enable 384 samples processing with <1 hour of hands on time. Equivalent call rates, percentage on-target, and coverage uniformity were observed between manual and automated processing across two different panels, over 6 runs with 192 samples. The expansion of available barcodes and automation of the AgriSeq library prep enables up to 1536 samples to be processed each day while reducing operator fatigue, the potential for technical errors, and the sequencing cost per sample.

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C. Buchanan-Wright, R.C. Willis, A.Burrell, M. Swimley, L. Baselgia, P. Siddavatam, R. Conrad

With advances in plant phenotyping approaches for quantitative genetic analysis and increasing complexity of gene pyramiding schemes, the number of markers required for successful molecular agricultural breeding programs is increasing. Historically, technology has been polarized between high-marker, high-cost microarrays or low-cost singleplex approaches that are not easily scalable. Targeted genotyping by sequencing is emerging as a powerful alternative for mid-density genotyping of 100s to thousands of markers in a high-throughput and cost-effective manner.  

We have applied AgriSeq targeted GBS, a high-throughput amplicon based sequencing workflow performed on the Ion S5 system to 3 economically important crops. A 2800 marker cucumber panel, an 1100 marker soy panel, and two independent corn panels targeting 900 and 1000 markers were designed for the AgriSeq workflow. The average genotyping marker call rate ranged between 91–98% for these panels, with >94% average uniformity and >99% on-target reads. >99.4% reproducibility has been demonstrated for this workflow over multiple independent library preps and sequencing runs, with genotype concordance to orthogonal array technologies of 99.4%. Compatible with high-throughput processing, the AgriSeq approach can multiplex up to 768 samples simultaneously and generate up to 1.6M genotypes per day. In addition, this approach allows for the discovery of additional SNPs and micro-haplotypes around the targeted markers which enable further traceability and association in downstream studies. These results demonstrate the utility of the AgriSeq targeted Genotyping by Sequencing for plant molecular breeding programs.

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Heather Koshinsky, John D. Curry, Maria Shin, Vineet Joshi

Genetic gain effectively relates four core factors that influence breeding progress: the degree of phenotypic variation present in a population, the probability that a trait will be transmitted from parent to offspring, the proportion of the population selected as parents for the next generation, and the length of time necessary to complete a cycle selection. The length of time is not only how many generations are required to complete a selection cycle, but also how quickly the generations can be completed. This includes the time taken to obtain genotypes from samples.

Targeted genotyping by sequencing is emerging as a valuable tool for high-throughput, low-cost single nucleotide polymorphism (SNP) detection in both plant and animal genomics. Plant breeding has a long history of integrating the latest innovations in biology and genetics to enhance crop improvement. Using Eureka Genotyping Panels developed for various plants, we have demonstrated genotypes in a day.

Aquaculture breeding programs need rapid, high-throughput genotyping to develop genetically improved stocks for cost-effective production. Older fish (more growing days), are often the larger fish. Thus, in aquaculture breeding the age of the fish can obscure genetic potential. Genotyping fish the same day they are spawned would increase the efficiency of the breeding program. Using a 500-plex Eureka Genotyping Panel developed for Oncorhynchus keta (chum salmon, a residual tetraploid), we have demonstrated genotypes in a day.

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