International Plant & Animal Genome Conference (PAG 30)

AgriSeq™ targeted genotyping by sequencing (GBS) has been utilized as a high-throughput and cost-effective genotyping solution for several economically important crops and animals. It is based on next-generation sequencing technology which can genotype various genetic markers.

In this study, we implemented AgriSeq™ to detect the adulteration of various raw or processed foods and other biological products. In total, 319 multiplexed markers representing 177 unique genera were designed to detect multiple species. The markers were designed on highly abundant genes present in mitochondrion and chloroplast in animals and plants respectively.

The performance of the AgriSeq™ panel was evaluated by analyzing 192 samples in a single run.

The samples were created by mixing DNA ranging from 7 ng to 1pg of 22 known species of domestic and wild mammals, birds, marines and freshwater fishes, invertebrates, and commercial crops. In addition, 32 samples of raw and processed commercial foods were also included. Sequence analysis indicated a wide range of coverage for the samples tested. Samples with 10 ng of DNA had one million reads while processed food samples had low number of reads. In addition, high concordances were observed among sample replicates and the mixed DNA samples. Results also indicate that the panel has high sensitivity as samples with a concentration as low as 1 pg of DNA were detected. Our high-quality data demonstrates that AgriSeq™ is a viable solution for genomic applications involving the analysis of 100s of multiple species in a single sequencing run.

For Research Use Only. Not for use in diagnostic procedures.

AgriSeq™ Targeted Genotyping by Sequencing is a valuable tool for high throughput, cost effective SNP detection and genotyping for Marker Assisted Selection or Breeding (MAS/MAB) in agriculture. AgriSeq™ is primarily being used for genotyping diploid organisms, leaving a segment of the plant genotyping, i.e., polyploids. Genotyping polyploids is more challenging compared to diploid organisms due to the presence of more than two sets of chromosomes and requires different analysis strategies.

We developed a statistical method to genotype polyploids using AgriSeq™. To evaluate the performance of the method we designed two AgriSeq™ panels for auto-tetraploid potato and auto-hexaploid chrysanthemum. Utilizing the AgriSeq™ HTS Library Kit, each panel was used to generate sequencing data from 95 DNA samples on the Ion S5™ sequencing system.

The results show that the mean genotype call rate for potato and chrysanthemum was 98.5% and 93% respectively. We evaluated the concordance for two cases: 1) Same sequencing data input; AgriSeq™ genotypes compared with fitpoly genotypes, 2) AgriSeq™ genotypes compared with genotypes from a microarray technology. For case 1, concordance was 99% and 96.5% and for case 2, concordance was 97.3% and 92.4% for potato and chrysanthemum respectively. In case 2, if we assume that the allele dosages differing by one are same then the concordance is > 99.5%.

The results demonstrate the performance of the genotyping method for polyploids to be used with AgriSeq™. The method is available as a plugin on Torrent Suite Software (TSS).

For Research Use Only. Not for use in diagnostic procedures.

 AgriSeq™ targeted GBS is being used as a high throughput and cost-effective genotyping solution in various applications from animal and plant breeding studies to parentage testing and genetic screening. One of the many features of this technology is supporting different type of markers including Single Nucleotide Polymorphisms (SNPs), Multiple Nucleotide Polymorphisms (MNPs), Insertions and Deletions (InDels), Long insertions and deletions (LongIndels) and other structural variants (SVs) (e.g. inversions, duplications). 

Structural variants are variants of size larger than 50bp and its primer design is slightly different than regular SNP design, having two amplicons per marker (one for wild-type and another for mutant). Because the current variant caller is incapable of making calls for such a marker, we developed an analysis method to genotype long indels and other structural variants, i.e., AgriSeqSV.  

The robustness of this technology has been demonstrated across 96 samples using 13 canine long indel markers (insertion, deletion, and inversion). Overall, 97% marker call rate across samples and 100% concordance were observed (when compared to truth data). It has been also shown that primer design and down-stream analysis were not impacted by the indel size.

High concordance across multiple samples with varying indel size indicates the reproducibility and flexibility of the method. Enhanced AgriSeq™ targeted GBS offers customers end to end solution for genotyping diverse marker types simultaneously using the analysis workflow. AgriSeqSV is available as a plugin on Torrent Suite Software (TSS).

For Research Use Only. Not for use in diagnostic procedures.

AgriSeq™ targeted Genotyping-By-Sequencing (GBS) has been used widely as high throughput and cost-effective genotyping solution in plant breeding to innovate and improve cultivars for several years. As the cultivated tomato, Solanum lycopersicum is the most popular garden crop, there is a growing interest in breeding cultivated and wild tomato varieties for selecting commercially important traits. The development of elite tomato cultivars highly resistant to diseases and pests from such breeding programs can give farmers excellent yields.

The success of an AgriSeq™ targeted GBS is dependent on having a high-quality and wellannotated reference genome. Since 2009 there have been four assemblies of the tomato genome released. Traditionally, we have gotten low design rates when using the most recent (SL3.0) reference genome and its associated dbSNP. This is mainly because tomato is diverse and the publicly available database has 71 million SNP which accounts for an average of one mutation for every 12 bases in its reference genome of size 850 Mb. Low-conserved regions with highly diverged sequences make primer placement very difficult in targeted genotyping.

Using publicly available databases that explored the genetic variation in tomatoes, we set out to create a custom dbSNP database. This database includes the intersection of all the mutations across 100 tomato genomes, resulting in about 2 million common, high-frequency SNPs. While it is impossible to account for every mutation (associated with thousands of cultivars), this approach has allowed the design of robust and high performing targeted GBS Tomato panels.

For Research Use Only. Not for use in diagnostic procedures.

Genotyping by Sequencing (GBS) is a robust tool in agrigenomics that is used to identify SNPs for a wide range of applications. High-throughput GBS requires cost-effective DNA extraction methods that produce consistent, high-quality results. However, current methods for genomic DNA extraction from plants yield highly variable results. The resulting final DNA quality can be tissue and even species dependent. Methods used for initial tissue grinding also heavily impact DNA yield and present a significant time drain when using traditional single-tube formats. Here we describe a high-throughput protocol in a 96-well-format for genomic DNA extraction that is suitable for use in targeted GBS applications. Our streamlined procedure is compatible with a range of plant tissues such as leaf punches, seed chips, seed embryos, and fruit. Tissues undergo mechanical disruption using stainless steel beads in a 96-well rack, and DNA is subsequently isolated using the MagMAX CORE AgGenomics DNA extraction kit along with Plant RNA Isolation Aid. The quality of nucleic acid extraction was first confirmed by qPCR, wherein preliminary testing determined that extracted samples did not contain significant carryover in the form of PCR inhibitors. Next, targeted GBS libraries were prepared by AgriSeq™ HTS Library Kit. Ion Torrent next-generation sequencing results revealed both high marker call rates and low sample drop-off. Our high-quality data demonstrates the value of our newly improved DNA extraction format as a higher-throughput, cost-effective complement to the AgriSeq™ Targeted GBS workflow.

For Research Use Only. Not for use in diagnostic procedures.

Targeted Genotyping by Sequencing (GBS) is a robust and cost-effective method for marker-assisted breeding and selection. Targeted GBS provides a scalable workflow, allowing for thousands of markers to be analyzed in up to thousands of samples per day.

The Applied Biosystems™ AgriSeq™ targeted GBS solution allows for up to 1536 uniquely barcoded samples to be processed in a single sequencing run. The current workflow utilizes a single-barcode approach, with 1536 barcodes available in 16 96-well plates. With amplicon coverage of 100X and using an Ion 540™ chip averaging 70M reads, up to 500 markers can be analyzed in 1536 samples on a single chip. Using an Ion 550™ chip averaging 115M reads, up to 750 markers can be analyzed in 1536 samples on a single chip. As GBS use expands, demand increases for higher throughput, faster turnaround, and increased multiplexing capability in GBS workflows.

Increasing available barcodes could increase the sample throughput of the AgriSeq™ workflow. In the current single barcode workflow, increasing available barcodes means increasing the number of 96-well plates to manage, which is undesirable. As an alternate solution for increasing throughput and barcode availability, we are exploring the use of dual barcodes. With this method, each sample would receive a combination of barcodes, allowing for more samples to be uniquely barcoded, which increases multiplexing capability and cost effectiveness without adding 96-well barcode plates. Here we report the feasibility of implementing the use of dual barcodes in the AgriSeq™ workflow and its implications for marker performance and coverage requirements.

For Research Use Only. Not for use in diagnostic procedures.

While the world population increases at an unprecedented rate, meeting the growing food needs continues to be a challenge. For more than a decade, the bovine dairy industry has employed the genetics of their cattle to improve production traits, such as milk yield and protein percentage. These methods have shown to be critical for the improvement of dairy cattle productivity. This breeding strategy is achieved by genotyping thousands of biallelic SNPs, interrogating loci well-distributed across the entire genome, potentially capturing all relevant quantitative trait loci (QTL), for use in Genomic Selection (GS). The application requires the interrogation of a fixed set of markers rapidly over thousands of samples, so medium-density, 25,000 to 100,000 marker microarrays are an ideal fit.

For genotyping dairy cattle, Thermo Fisher Scientific provides numerous Applied Biosystems Axiom microarrays measuring around 65,000 markers. These arrays, such as the Axiom Bovine Genotyping v3 Array includes 44,000 markers recognized by the Council on Dairy Cattle Breeding (CDCB). Recently, the CDCB released a list of 80,000 markers used for genetic evaluation.

Thermo Fisher Scientific has developed a 100,000-marker microarray to interrogate all 80,000 CDCB relevant makers. In addition, this array includes markers for even genomic coverage, economically valuable traits-associated markers, sex-linked markers, microsatellite imputation markers, and parentage verification, such as the International Society for Animal Genetics (ISAG) 200 and ICAR 354 markers. This higher-density panel can also be useful in tracking undesirable genetic trends, such as inbreeding depression, to drive overall genetic improvement of dairy cattle in commercial breeding programs.

For Research Use Only. Not for use in diagnostic procedures