To Reduce Complexity and Cost
PGT’s sample tagging technology relies on the labelling of individual test subjects and loci or genes within the test subjects’ genome with a uniquely identifiable DNA tag which is maintained during subsequent manipulations. Following sample preparation in this way, the samples are pooled and can be selected to be limited to particular regions of interest before the pool populations are analyzed using NGS. Individuals and subsets loci or genes across the population of pool samples can be computationally grouped based upon the sequence used to tag them. Thus this technology allows for multiplexing, the processing of a large number of samples in parallel on a high-throughput instrument. Sample multiplexing is an especially useful technique when targeting specific genomic regions or working with smaller genomes. It enables, the rapid analysis of large sample numbers during a single experiment. Importantly, it is cost effective, improving productivity by reducing hands on labour, time and reagent use; the pooling means that region of interest enrichment can be done once for the pool. rather than separately for each of the samples in the pool.
PGT’s sample and molecule tagging technology features include:
- Scalability (high throughput at low cost) by obtaining sequence data to determine genotype of multiple genes from multiple samples. This enables the pooling together of small samples to use the multi-Gb capability of most NGS platforms cost effectively.
- Focus on regions of interest (pulling out sequences to a given loci or gene from the whole population of samples and linking the data back to each original test sample). In one implementation, this is accomplished by amplifying one or more fragments by PCR, using a gene-specific primer and a tag-specific primer. The amplicons will contain sequence from the target gene and the tag, so that the sample can be identified. An typical implementation utilizes hybridization-based pull-out of the regions of interest using large numbers of synthetic oligos which bind to and then select the genomic regions to which the oligos specifically bind.
Examples of Issued US Intellectual Property
“Methods and compositions for tagging and identifying polynucleotides” US issued patent 7,393,665
“Identifying fragments corresponding to (one or more) specific genes, and simultaneously genotyping multiple samples at multiple loci” US issued patent 8,148,068
“Identifying fragments corresponding to (one or more) specific genes, and sequencing the fragment (obtaining tag and fragment sequence).” US issued patent 8,168,385
“A method of sequencing multiple fragments to obtain sequence of the tag and of the fragment, using a sequencing primer corresponding to sequence present in the tag.” US issued patent 8,318,433 B2
Sequencing pooled cDNA, obtaining sequence from fragment and tag to obtain information about gene and sample from which each fragment originates. US issued patent 9,194,001
Amplifying fragments corresponding to a specific gene, and quantifying the number of unique tags present in the amplified population. US issued patent 8,476,018
Amplifying one or more fragments by PCR, using a gene-specific primer and a tag-specific primer. Tag primer using a conserved region so will amplify from pooled population. The amplicons will contain sequence from the target gene and the tag, so that the sample can be identified. US issued patent 9,018,365
Using a microarray to sort the pooled fragments according to their tags. Probes on the array correspond to the tag sequence. Thus fragments from each original sample are each hybridised to a specific tag. US issued patent 8,470,996