What is Haplotyping?

1
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

2
Three Underlying Technologies
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
3
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.
4
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.

5
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