By Dusty Vyas, Senior Scientist at LGC Biosearch Technologies
Targeted GBS
The application of targeted genotyping by sequencing (targeted GBS) in marker assisted selection (MAS) and genomics assisted breeding (GAB) has made irrefutable impacts in transforming selection strategies in applied breeding objectives. The continued rapid advancement of next-generation sequencing technology to produce dense genome-wide SNP markers, coupled with its substantial cost reduction for genotyping in several crops, makes targeted GBS a must-implement method in most breeding programs1.
Increasing cost and time efficiency in the application of targeted GBS in high throughput (HTP) genotyping workflows has driven developments in available chemistries and HTP solutions such as original Amp-Seq and Amp-Seq One. The implementation of original Amp-Seq effectively into standard workflows validates these developments and is essential in driving forward strategies in breeding programmes for the whole breeding community. Standard end-point genotyping workflows could be upgraded or superseded to provide 3000-times more data points (3000 SNP panel) in the same time frame. This increase in data points could change the perspective, vision and selection process in breeding programs.
Amp-Seq is a midplex targeted GBS approach (up to 5,000 targets) that comprises high quality panel designs, simplistic workflows with maximum data generation (including minor allele frequency information), reduction of sequencing costs of up to 50% and considerations to environmental impacts (ambient temperature stable components and minimal dry ice shipping).
Installing a new workflow
Serious considerations are taken when laboratories are looking to improve workflows not just from a cost efficiency perspective. The success of implementation and the support that is received plays a key role in the selection process of evaluating potential suppliers. Robust supply chain of reagents, onsite and offsite support reduces the stress and challenges faced during the re-evaluation process. When help is needed, the expectation is that suppliers will respond and support in timeframes that are critical in a time-pressured environment.
When installing Amp-Seq into your laboratory, there is a worldclass installation team backed by a robust supply chain that will ensure that the workflow is successfully implemented and delivers to your expectations. The team are also on hand to resolve any future issues post-installation, should the need arise.
This collaborative approach to working with customers extends to panel development and custom implementation into workflows. This is a major part of how LGC Biosearch Technologies ensures success for customers in increasing throughput for their individual breeding directives.
Panel design
Panel design is the foremost critical aspect in the application of any targeted GBS strategy. Biosearch Technologies not only works iteratively with customers on the SNP, indel and MNP (multiple nucleotide polymorphism) targets to maximise success potential of the developed Amp-Seq panel but we also validate the panels through rigorous testing in our own laboratories prior to validation in our customer’s facilities and process flows.
How Benson Hill did it 
A collaboration that has seen a step change in increasing capability without compromise of data quality was with Benson Hill. Benson Hill is an innovative seed company that is at the cutting edge of driving efficiencies in genotyping screening approaches for soy variety development “for better feed, food and fuel”. Originally running Amp-Seq, then implementing Amp-Seq One, the teams’ feedback on the effective application was very positive.
Teamwork extended to onsite workflow assessment, optimisation and solutions to fine tune the validated Amp-Seq soy panel in established Benson Hill process flows. This ensured that the team could move into full production scale screening, which is critical for HTP targeted GBS genotyping chemistries, rapid turnaround times for sequencing data generation and ultimately selection of lines to move forward.
Industrial genotyping workflow
Pragmatic HTP workflows for the assessment of accurate genomic estimated breeding values (GEBV’s) in ultra-high and imputed mid-density training populations with increased accuracy in phenotypic measurements do maximise the possibility of not only the accurate selection earlier in crossing programmes, but also enable utilisation of transcriptomics, metabolomics and AI driven understanding of key traits contributing to an overall improvement in genetic gain.
Figure 1. Schematic of Amp-Seq One process. The Amp-Seq One workflow reduces the total time required to complete the library preparation by over 1 hour and doubles throughput versus 2-stage methods. This simplified workflow with one PCR stage and reduced numbers of reagents halves the hands-on time.
Amp-Seq provides a straightforward approach to the production of sequencing libraries through input of lower DNA concentrations into standardised PCR cycling, provision of dried down index plate (availability >36K indices) and magnetic-based cleanup protocols, in standard working hours (figure 1). This, combined with HTP instrumentation, enables not only cost-efficient application of targeted GBS but also screening in breeding programmes for expediated variety development from GWAS to QC evaluation with marker densities as low as 100 SNP targets.
Superior sequencing data is essential to maximise association with targeted traits, foreground and background markers in crosses. Even small incremental improvements can save time and costs in varietal development. The release of Amp-Seq One has maintained high quality data seen with original Amp-Seq with the benefits of reduction in labour costs and throughput. Data from a soy 1,000+ panel run on a diverse sample set (figure 2) saw comparable performance for both technologies, including reads mapped (%) and on target calls (%).
Figure 2. Dataset for 1,100 marker panel for soy, illustrating almost identical performance between Amp-Seq One and original Amp-Seq. DNA was purified using the sbeadex Plant kit (n=384). Sequencing depth 200X.
The rapid development of Amp-Seq One underlines the commitment of Biosearch Technologies to understand, develop and provide solutions to the breeding community. Step changes in approaches for the application of genomic technologies like Amp-Seq One are game changers that re-think how NGS can be applied in breeding programmes to maintain impetus for rapidly changing climate and environmental challenges.
Further reading
- Amp-Seq One app note
- Amp-Seq One data sheet
- Original Amp-Seq app note
- Blog: A guide to selecting the optimum trait mapping genotyping tool within your budget
References
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Molecular Plant 17, 552–578, April 1 2024. Admas Alemu et al. Genomic selection in plant breeding: Key factors shaping two decades of progress.
