Nanopore Haplotyping

Nanopore Haplotyping

Why are new methods for SNP detection and haplotyping desirable?

Genotyping and a better understanding of human genetic variation will profoundly affect our understanding of disease, accelerate the rate at which new drugs are brought to market (pharmacogenetics) and improve patient care using existing pharmaceuticals (diagnostics and "personalized medicine"). While genotyping using single nucleotide polymorphism (SNP) markers, together with attempts to relate these markers to an observed phenotype or clinical response, have become the method of choice for performing disease association studies, several studies suggest that individual SNPs may have poor predictive power, either as pharmacogenic loci or as tools in human health research and care. On the other hand, haplotypes can correlate a specific phenotype with a specific gene in a small population sample even when individual SNPs cannot . Such results suggest that the phasing of multiple SNPs along a single chromosome -- the haplotype -- would better predict physiological response.

How can a nanopore determine a haplotype?

We are developing the technology and basic science needed to use nanopores for high speed SNP detection and haplotyping. The development of this new method takes advantage of four emerging discoveries: (1) A membrane channel, or nanopore, can be used as a high-throughput device that detects and probes the full length of a DNA molecule as it translocates through the nanopore; (2) Improved approaches to molecular engineering of high affinity, high specificity zinc finger proteins make it possible to label just about any targeted DNA sequence in an unrestricted and comprehensive fashion; (3) The ionic current through a nanopore is sensitive to local changes in the cross-sectional area of a translocating polymer molecule; (4) Ion beam sculpting is a planar fabrication method that allows us to create nanoscale pores with dimensions that can probe double stranded DNA. Our work will optimize the minimum fragment length and number of different zinc-finger proteins needed to achieve reliable SNP identification and high-speed haplotyping.

The figure above explains the steps of nanopore haplotyping. (1) ZFPs are incubated with DNA of unknown haplotype. Depending on which allele exists at each SNP location (colored regions of the unknown DNA), ZFPs either bind or do not bind. (2) After incubation, the DNA with any bound ZFPs are translocated through a nanopore. (3) Each ZFP-labeled position on the DNA gives rise to a distinctive electrical blockade signal as the DNA translocates through a nanopore.

Because a nanopore can detect and "read" single molecules, a major advantage of the proposed method is that there is no need to amplify the target molecules. Thus, the haplotype of a genomic sample can be directly determined. Although a statistical sampling will be needed to establish a high degree of confidence in the measurement, it is likely that this will require the measurement of no more than 200 target molecules. This corresponds to about 500 picograms of human genomic material, which can be directly obtained using standard sampling methods.

It will probably be evident to the reader that if one had a method to distinguish and identify each of many different sequence fragments in a single sample, a single nanopore could genotype a mixture containing multiple different DNA sequence fragments each of which would contain different SNPs. Similarly, it will also be evident that since very long strands of DNA can be translocated through a nanopore, it should be possible to use a single nanopore to identify the allele present at multiple different SNP sites along the length of such a strand (haplotyping) since the instantaneous position along the DNA's length will be measured by the current block duration. Finally, combining these concepts, it will be clear that complex mixtures of different fragments, even random-length fragments, could also be analyzed using this method.