ddRAD genotyping

Less is more

One of the major benefits of low error rate provided by Illumina platform is their utilization for accurate SNP calling. However, for large scale experiments where cost-effectiveness has to cope with massive panel of samples (breeding, ecology, association-studies, etc.), whole genome sequencing remains an unaffordable option. One of the most effective techniques in the cutting down of such cost is to reduce the genome to a fraction of its size before sequencing. 

The method of choice adopted by IGATech for genotyping-by-sequencing is the so-called ddRAD (double digest Restriction Associated DNA). The system provides complexity reduction by selecting genomic DNA fragments generated by cleavage by both restriction enzymes, one per side. This has the advantage of having each genomic locus characterized by a specific size in the library of fragments. Adopting in silico simulation it is possible to fine-tune the amount of genomic loci to be selected. 

This capability allows to accommodate the adequate sequencing yield to obtain effective coverage to most of loci and thus providing high and reproducible genotyping ratio across individuals also in large cohorts. Moreover, compared to array-based solutions, sequencing is an “open system”, meaning that genetic variability is sampled unbiasedly: this has a critical impact on experiments involving genetic backgrounds and haplotypes not exhaustively represented by the panel of markers used in an array or any previous experiment.

Expertise

IGATech has successfully conducted many genotyping-by-sequencing experiments on both plants and animals, with genome size ranging from hundreds of megabases to tens of gigabases. Despite such very big differences in genome size and complexity we managed to obtain effective complexity reduction and to have more than 95% of sequencing data in loci with adequate coverage overall. This why our internally developed protocol is also suitable for the finding of discriminatory marker for varietal identification and contamination levels estimation also in food substrates. Our lab practices are devoted to obtain reproducible experiments and our bioinformatics analyses to obtain high quality data points. Our internal barcoding system allows reaching a virtually unlimited multiplexing. We provide tailored consultancy for every project to make sure the outcome will meet our customer expectations. Every genome and experimental design have its challenges.