What is Haplotyping? T C A G Diploid organisms may be polyallelic at some loci (highlighted to the left) A C C G SNP sequencing would show the genotype as A/T, C, A/C, G This would still leave two possibilities for the true haplotype: ACCG/TCAG or ACAG/TCCG. Single-molecule haplotyping can distinguish between chromosomes, and tell us that the true haplotype is ACCG/TCAG Haplotypes give more information, and have better predictive power than SNP sequences

Three Underlying Technologies ACCTGTCAGGCGTACCA TGGACAGTCCGCATGGT Padlock probe labeling is used to allele-specifically label the SNPs of interest. Molecular combing is used to stretch the DNA molecules on a surface prior to imaging FIONA is used to localize the labels. Combining all three gives barcoded DNA

Molecular Haplotyping Why? Diploid organisms (e.g. humans) may be heterozygous Knowing the correlations of SNPs on each individual chromosome (the haplotype) confers more predictive power than SNPs alone Most genotyping techniques use bulk PCR products, and cannot distinguish chromosomes Single molecule analysis can allow us to distinguish discrete populations How? First, DNA is allele specifically labeled with single fluorescent probes using padlock-probe labeling Second, DNA is stretched onto a surface using molecular combing DNA backbone and fluorescent nucleotide labels are imaged using FIONA Double stranded DNA can be stretched with molecular combing onto a surface, as shown. Single stranded DNA can potentially be stretched if it is stabilized with RecA, a single-stranded DNA binding protein. Labeling of ssDNA with padlock probes can be more efficient with ssDNA.

Haplotyping and Genomics Current genotyping technologies work on bulk PCR products, and hence cannot distinguish heterozygous haplotypes, because they cannot distinguish products from different chromosomes Haplotypes have much more predictive power than simple SNP sequencing for determining disease susceptibility and drug interactions Once general patterns have been discerned using genome-wide SNP scans, haplotyping will be the dominant means of determining precise correlations between regions of genomic interest and disease. We will focus on a 500 kb region containing the HOXA locus on human chromosome 7, as part of the ongoing work of the International HapMap consortium.

Single Molecule Fluorescence Haplotyping 3 kbp 17 kbp In our approach, PCR products are site- and allele-specifically labeled with single dye molecules, and are imaged to establish a “barcode” which can be used to determine the haplotype at selected SNPs. Different alleles are labeled with different dyes (e.g. Cy3, Cy5), and can be distinguished by color