Less is more
We were able to make an excellent map to anchor our genome assembly. IGATech support was very helpful.
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 via restriction enzymes. However, when genome complexity reduction is not accurately tailored on specific genome size, repeat content and nucleotide composition, it can generate drops in the genotyping ratio of valuable and robust markers thus reducing the overall cost-effectiveness.
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 then possible to fine-tune the amount of genomic loci to be selected.
Genetic variability is sampled unbiasedly
This capability allows to accommodate the adequate sequencing yield to obtain effective coverage to most of loci and thus providing i) robust heterozygous/homozygous discrimination and ii) high 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.
Over the past years we came to heavily rely on the excellent services provided by IGATech, whose expertise, efficiency, helpful and custom-oriented attitude accompanied us in our scientific growth in the field of genomics. A recent service extension is their double enzyme ddRAD Seq approach we are currently using with great success in several crop species.
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.
- Ascertain bias-free experiment
- Discover and genotype diversity in one experiments
- Competitive prices
- Up to 768 sample in a single Illumina lane
- Scalability of markers yield from thousands to hundreds of thousands
For every project we want to make sure that the outcome will meet our customer expectations.
- DNA normalization and purification
- Library preparation and QCs
- Sequencing (paired-end 125bp or single-end 150bp)
- Data analysis from raw data to ready-to-use genotype scoring tables
- Complementary analyses:
- linkage analyses / QTL mapping
- association studies
- molecular barcoding / varietal identification
- diversity / population structure