What is Genotyping by Sequencing?

Genotyping by Sequencing, also known as GBS sequencing, is a powerful genetic screening method. Molecular breeding utilizes DNA markers (SNPs) that are tightly linked to phenotypic traits to assist in selection to deliver improved plant and animal species for agricultural use. As major drivers like population growth, urbanization and economic development feed into an ever-complex agricultural landscape, agricultural genomics has taken a central role to offer sustainable solutions for the global food challenge.

Among the many genotyping by sequencing platforms, such as PCR- and array-based methods, Next-Generation Sequencing (NGS) has proven to be an exceptional tool for the discovery, validation, and assessment of genetic markers. NGS offers high multiplex levels, robust call rates, and an economical approach to genotyping large numbers of SNPs and samples. Targeted genotyping by sequencing (GBS) can focus on thousands of markers to provide a cost-effective, high-throughput genotyping solution that is customizable, flexible, and scalable.

Some of the key genotyping by sequencing plant (plant genotyping) and animal molecular breeding applications and genotyping methods include:

  • Marker-assisted selection (MAS)
  • Genomic selection (GS)
  • Trait mapping
  • Quantitative trait locus (QTL) screening
  • Marker-assisted backcrossing (MABC)

AgriType 2.0 Solution

Cost-effective targeted genotyping solution to accelerate molecular breeding

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AgriType® Targeted Genotyping by Sequencing Solution

Cost-Effective Solution to Accelerate Molecular Breeding

The AgriType Targeted Genotyping by Sequencing Solution is designed and developed to address large-scale genotyping projects in agrigenomics (the application of genomics in the field of agriculture). The AgriType solution is powered by an advanced primer design algorithm and an efficient ultra-high multiplex PCR-based target enrichment chemistry. AgriType genotyping by sequencing provides a cost-effective solution to construct NGS libraries to quickly and efficiently screen hundreds to tens of thousands of markers of interest to accelerate genotyping studies in fields such as aquaculture, livestock farming, and seed breeding.

  • Scalable High-Throughput Genotyping System. Target thousands of relevant markers in the same assay.
  • Fast Turnaround Time. Get custom assays delivered in 4 to 6 weeks. Use AgriType® genotyping solution to accelerate your large-scale molecular breeding programs. Our PhD-level expert scientists are here to provide speedy and comprehensive support throughout the design, ordering, and technology adoption process, including excellent after-sales technical support.
  • Cost-Effective Solution. Process thousands of samples at a time on Illumina® platform.
  • Minimizes consumable consumption. This single-plate workflow reduces consumable cost and loss of material from material transfer.
  • Streamlined, Automation-Friendly Workflow. Generate sequencing-ready libraries in just 3 hours using a simple, three-step protocol that can be easily automated.
  • High-Quality, Reproducible Results. Prepare high-quality NGS libraries with high marker call rates and efficient use of sequencing reads.

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High Concordance with Whole Genome Sequencing Data

A 2,000+ amplicon AgriType custom NGS panel for plant breeding was developed for a large crop science company. To test the robustness of AgriType solution, 8 samples that were previously genotyped using whole genome sequencing (WGS) were used to construct AgriType target-enriched NGS libraries and the data compared. Of the 2,000+ markers tested, 99.5% showed high concordance with WGS results, and perfect concordance was obtained at the sample level.

Threshold% Markers% Samples
>95% Concordant99.5%100%
>80% Concordant99.7%100%
<80% Concordant0.3%0%

“We finally ran our pilot. The data is beautiful! Targets had great coverage and it was very uniform. The protocol is well-written and easy to follow. I’m not sure how Paragon Genomics does it but it is really impressive. Thanks so much to you and your custom panel design team! They did an amazing job! SUPER great experience.” - Scientist, Fortune 500 Agricultural Chemical Company

Streamlined and high-throughput targeted genotyping by sequencing solution

The AgriType® 2.0 genotyping by sequencing solution has been optimized to accommodate higher-throughput needs and offers a streamlined workflow that is easily automated in both 96 and 384 well plates. The multiplex PCR-based protocol can be completed in just 3 hours, with very little hands-on-time of 25 minutes via a simple multiplexing PCR followed by an indexing PCR.

DNA Purification and QC

1.Multiplex PCR

2. Dilution Step

3. Indexing PCR

Quality Control

Sequencing

Data Analysis

AgriType 2.0® Target Enrichment and Library Preparation

3-5 hours of total assay time, 20 minutes of hands-on time

High-Quality AgriType NGS Panels for All Panel Sizes

AgriType® panels show consistently superior sequencing specifications, accomodating a wide gamut of panel size, to meet all your research needs. AgriType 2.0 offers detection of up to 7000 targets in a single panel.

Amplicons
per
reaction		Uniformity
(0.2x Mean
Coverage)		% Mapping Rate% On-Target Rate
15100%98.8%98.9%
109100%96.2%99.2%
20099.5%97.2%96.8%
60099.5%99.0%97.5%
150098.8%99.7%96.4%
Panel SizeUniformity (%)Mean Call Rate% On Target Rate
2550 (Cotton)95%97%92%
436 (Tomato)96%98%99%
6808 (Soybean)90%95%92%

Genotyping By Sequencing: Frequently Asked Questions

Why sequence in agriculture? What is its impact?

High throughput phenotyping and genotyping offer critical decision-making information to feed breeding strategies. The growth in genomic technology has allowed researchers to accurately assess genetic markers with high resolution and link them to phenotypic variation. This has led to the identification of certain markers as relevant for drought tolerance, response to a particular virus, etc.

What are the differences between genotyping with NGS vs microarray?

Array-based approaches to genotyping have historically been the gold standard to analyze regions of the genome for agriculture. However, with the advent and massive development in next-generation sequencing (NGS), researchers have turned to genotyping by sequencing as a method to screen novel regions of interest for plants and animals. For applications such as genetic mapping and variant discovery, genotyping by NGS offers a lower cost per sample.

What do you need to get started on a custom panel design?

We only require the targeted bases such as SNPs you’re interested in and the reference genome for the crop of interest. The bases can be provided as a BED file and submitted to our design portal (https://www.paragongenomics.com/panel-design-my-design-requests/)

How many samples can I sequence at once?

AgriType uses a powerful multiplexed approach that allows users to interrogate large regions of interest across hundreds to thousands of samples. We support 2688 Illumina combinatorial indexes for high-throughput workflows. Our high uniformity of amplification, high mapping rates, and on-target rates enable efficient use of sequencing space. For example, for a 1000 amplicon panel, up to 150 samples can be fit on a Miseq and 1300 samples on a NextSeq with an average of 100X coverage.

How do I generate genotypes with the sequencing data?

AgriType libraries are compatible for sequencing on major sequencing platforms such as Illumina® and Ion Torrent™. Downstream data analysis can be completed easily on the Illumina basespace variant callers or Torrent space and add on modules to enable labs to assess relevant sequencing metrics. If relevant, open source bioinformatics pipelines like TASSEL-GBS can be utilized for processing of raw GBS sequencing data into SNP genotypes.
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