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Advantages over microarrays!!! Completely internally controlled No tissue heterogeneity

Goal: Precise measurement of cis- phenotypic variabilty with respect to transcript levels. Strategy: SNP -based quantitation of allelic transcript ratios in heterozygotes. Advantages over microarrays!!! Completely internally controlled No tissue heterogeneity

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Advantages over microarrays!!! Completely internally controlled No tissue heterogeneity

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  1. Goal:Precise measurement of cis-phenotypic variabilty with respect to transcript levels.Strategy:SNP-based quantitation of allelic transcript ratios in heterozygotes. Advantages over microarrays!!! • Completely internally controlled • No tissue heterogeneity • Isolates cis effects from trans effects

  2. Rob Mitra

  3. Allele quantitation via polony-based single-base extension • Isolate mRNA • External RT-PCR • Standardized Polony Amplification • Single Base Extension Gene-Specific ~600 bp Common Primers

  4. Allele quantitation via polony-based single-base extension Count ratios & look for deviations from 50:50 (standard binomial statistics) 180 spots, 33:66 ratio -> p = 4e-06 500 spots, 40:60 ratio -> p = 4e-06 2000 spots, 45:55 ratio -> p = 4e-06

  5. Mouse Inbred Strain Haplotypes / Linkage Disequilibrium?(Fibulin 2) A/J DBA/2J 129/SvJ C G A A C C T G C A T G G G C A T T A T G G G C A T T 54 857 878 1162 1622 Genomic distance =~ 60 kb 1759 2378 2825 4336

  6. A = load_image(Z); A1 = image_subsection(A,X1,X2,Y1,Y2); A2 = wiener2(A1, [5 5]); A3 = medfilt2(A2, [5 5]); A4 = imadjust(A3, stretchlim(A3), [0 1]); A5 = imopen(A4, strel('disk',DW)); A6 = imsubtract(A4,A5); A7 = imextendedmax(A6, 10000); [Xlabeled, Xnumobjects] = bwlabel(A7,4); Matlab Image Analysis

  7. Polony-based analysis of 7 transcribed SNPs in mRNA from an (A/J x DBA/2J) F1 pup • 3 out of 7 deviate from 50:50!!

  8. Nonsense Mediated Decay Single-base pair deletion at base 2092 of Junctophilin 3 leads to frameshift in coding region

  9. Potential Questions / Directions • Can the intersection of the mouse SNP database and HUMMUS/regulatory elements be used to predict heterozygosities that will lead to allelic skewing? • Fraction of transcripts with significant deviation from 50-50 in a single individual? • Within a population, are specific transcripts “looser” than others with respect to variation of expression? • Individual-to-individual variation in a given non-50-50 allelic ratio (assuming identical genetic background)?   • Does choice-of-tissue matter? How often? Need we bother? • Can we find instances where haplotype clearly matters more than individual SNPs? • Can quantitative models be defined around how specific regulatory SNPs impact transcript levels?   • Can we define the full complement of cis-heterozygosities of single locus with respect to haplotypes, transcript levels, and protein levels?

  10. Potential Questions / Directions • Stochastic Monoallelic Expression • Partial or Complete Imprinting • Nonsense-mediated Decay & Common Haploinsufficiencies • X-inactivation • Loss of Heterozygosity • Somatic Mosaicism 

  11. Add single Cy5-dNTP • Scan • Photocleave Cy5 group Polony Sequencing (Rob Mitra) Is there a robust algorithm for rapid image alignment? What is the minimum number of pixels necessary to call a polony sequence correctly? Can image analysis for polony sequencing be automated? (Rob Mitra)

  12. GTGT AGTGT AGTGC CAGCGA CACACA (error signatures)

  13. (1) Image Acquisition (2) Image Alignment (3) Pixel-by-Pixel Sequencing (4) Polony Sequence Calling

  14. Polony Sequencing Software • Rapid Image Alignment to error of < 0.5 pixels • Sequence calling is highly accurate even at single-pixel resolution • Polony Signature Object algorithm correctly calls 23/23 polony sequences (total of 84 bases)

  15. Future Directions for Polony Sequencing • Collaborate with Rob & George on… • Further developments in sequencing software • Longer sequence reads with new protocols • SAGE Library Sequencing as tangible short-term goal? • Alteration of nucleotide chemistry • Sequencing at higher polony densities • Fiber-Fish Polony Sequencing

  16. Acknowledgements • George Church • Ting Wu • Matthew Meyerson & Lab • Debbie Nickerson & Lab • Xiaoping Lian (Xeotron) • Andy Bhattacharjee (Agilent) • Rob Mitra • Ben Williams • Greg Porreca • Jun Zhu • Vincent Butty • Dennis Vitkup • John Aach • The Church Lab

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