Full control of your experiment
Either choosing hybridization-based systems or primer extension, the sequencing information will always provide additonal and off-target data, eliminating errors in diversity estimation caused by closed genotyping experiments as in arrays.
Several scenarios and specific analyses require moving from random markers (for which ddRAD is our system of choice) to targeted regions when performing genotyping experiments. Focusing on a specific set of SNPs, genes or genomic locations is a crucial factor to cope with tasks such as genomic selection, local adaptation studies, TILLING, eco-TILLING, fine mapping, GWAS, genomic selection and many others. IGATech have optimized protocols for both hybridization-based and primer-based enrichment procedures.
Single primer enrichment technology - Allegro
IGATech, in collaboration with Nugen inc., have implemented a targeted genotyping system based on single primer enrichment technology (Allegro, Tecan Genomics), which provides a very efficient approach to specifically target SNPs of interest. Every enriched read will be useful to the genotype calling of one selected SNP site. Nonetheless the technology allows to discover novel alleles by leveraging the sequencing information ensuring no restriction of transferability of each panel to other populations or even species. Sequencing read length can be scaled depending on the need of unknown (rare) variants versus the cost-effectiveness of focused genotyping.
Allegro provides highest reproducibility in the sequencing-based genotyping panorama while maintaining flexibility of continuous customization.
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Read our seminal paper (Annals of Botany)
HYBRIDIZATION-BASED (Sequence CAPTURE)
Utilization of hybridization-based enrichments have several advantages. They can be used also when a reference genome/transcriptome is only available for closely related species (or even genera) or when no information on polymorpisms and rate of occurrence is available. The technique is resilient to mutation rates and therefore prevent allelic-specific enrichments. Hybridization-based enrichment is the best suited approach for discovery approaches on gene panels/families or large genomic loci, given the possibility to yield a wide target with uniform coverage. IGATech has developed in-house 384-plex UDI (unique dual index) to cope with large scale experiments on NovaSeq6000 platform. The target region can vary from few Mbp up to 200Mbp.
For small amounts of loci (tens up to hundred) in very large scale experiments (thousands of samples) IGATech can deploy a customized multiplex PCR panel to run massive genotyping based on simple amplicon sequencing, providing a high-throughput and cost-effective platform. The panel can be easily modified and increased in markers over the time without an impact on costs.