Varietal Counting Of Nucleic Acids For Obtaining Genomic Copy Number Information

What is this patent about?

’589 is related to the field of genomic analysis , specifically methods for determining the copy number of DNA or RNA sequences in a sample. Traditional methods like whole genome amplification (WGA) introduce biases due to non-uniform amplification, leading to inaccurate copy number estimations. This is particularly problematic when analyzing small amounts of DNA, such as from single cells, where stochastic effects are amplified.

The underlying idea behind ’589 is to tag individual nucleic acid molecules with unique identifiers (tags) before amplification. By counting the number of unique tags associated with a particular genomic location after amplification and sequencing, the original copy number can be accurately determined, minimizing the impact of amplification bias. This approach essentially converts sequencing into a counting method, where each unique tag represents a single original molecule.

The claims of ’589 focus on a method comprising obtaining segments of genomic nucleic acids, tagging these segments with unique nucleic acid tags, amplifying the tagged molecules using PCR, sequencing the amplified products to generate tag-associated sequence reads, mapping these reads to a reference genome, and then counting the number of unique tags assigned to each location on the genome. This process allows for the determination of genomic copy number information that is less susceptible to amplification distortion.

In practice, the method involves fragmenting genomic DNA or cDNA, attaching unique tags to these fragments, and then amplifying the tagged fragments using PCR. The resulting amplified DNA is then sequenced using high-throughput sequencing technologies. The sequence reads are mapped back to a reference genome or cDNA library, and the number of unique tags associated with each genomic location is counted. This count provides an estimate of the original copy number of that region, effectively correcting for any amplification biases introduced during PCR.

This approach differs from prior methods that rely on uniform amplification of the entire genome. By tagging individual molecules before amplification, ’589 allows for accurate copy number determination even when amplification is non-uniform. The use of unique tags enables the differentiation of PCR duplicates from truly independent molecules, providing a more accurate representation of the original sample's genomic content. This is particularly useful for analyzing single cells or other samples with limited amounts of DNA or RNA.