Preparing samples for Eureka Genomics’ Mass Genotyping by Sequencing Technology methodology starts with heating DNA to melt it apart and break it into smaller pieces, “which makes the hybridization of the next set of probes onto it much easier,” explains John Curry, Ph.D., senior scientist.
For each single nucleotide polymorphism (SNP) to be interrogated three barcoded probes are then added: a phosphorylated right hybridization sequence, and two left hybridization sequences, which differ by a complementary SNP and permit discrimination between the two different alleles in the genomic sequence.
The hybridized probes are then ligated and act as a template for the subsequent PCR reaction, which further adds sample specific indexes.
The assays (one sample, but hundreds of loci, per well) begin in 384-well plates and are combined and spun down into a small library “so we’re really taking a few milliliters of PCR products and reducing it down to 100 µL of library,” explains Dr. Curry. “And a portion of that library goes into the sequencer.”
This type of ligase discrimination for SNPs has been done for 20 years. Yet “whereas before people would do this assay one at a time, or 40 at a time, and resolve it on a PAGE gel, we’re resolving it on a next-gen sequencing platform,” he continues.
“So we’re able to put thousands of samples, with hundreds of loci, into a single tube, onto a single lane of an instrument, and get the information back, and decipher it, and determine the genotypes for basically 1,000 x 100 SNP-sample combinations.”
The assay never actually has to read the biological information itself “because they’re all on the probes that are designed from the biological information,” notes Dr Curry. This allows for shorter, more economical reads. In addition the barcodes can be multiplexed.
“We’re able to drive the cost down to fractions of a cent per animal SNP-combination, and do them all at once.” Eureka Genomics has already commercialized this process for agricultural and clinical applications.