1 / 38

Genotyping and Genetic Maps Bas Heijmans Leiden University Medical Centre The Netherlands

Genotyping and Genetic Maps Bas Heijmans Leiden University Medical Centre The Netherlands. Pedigree file in linkage format. 1 1 1 1 2 2 2 2 2. 1 2 3 4 1 2 3 4 5. 0 0 1 1 0 0 1 1 1. 0 0 2 2 0 0 2 2 2. 1 2 2 1 1 2 1 2 1. 1 1 1 1 1 1 1 1 1. 1

orpah
Download Presentation

Genotyping and Genetic Maps Bas Heijmans Leiden University Medical Centre The Netherlands

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Genotyping and Genetic Maps Bas Heijmans Leiden University Medical Centre The Netherlands

  2. Pedigree file in linkage format 1 1 1 1 2 2 2 2 2 1 2 3 4 1 2 3 4 5 0 0 1 1 0 0 1 1 1 0 0 2 2 0 0 2 2 2 1 2 2 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 3 2 2 0 0 5 6 5 2 4 3 4 0 0 8 7 7

  3. Pedigree file in linkage format marker data (1 marker) disease status person id family id mother father sex 1 1 1 1 2 2 2 2 2 1 2 3 4 1 2 3 4 5 0 0 1 1 0 0 1 1 1 0 0 2 2 0 0 2 2 2 1 2 2 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 3 2 2 0 0 5 6 5 2 4 3 4 0 0 8 7 7

  4. Marker choice for genome-wide linkage scans • Short tandem repeats (STR, a.k.a. microsatellites) because: • High heterozygosity (1 STR ~ 5 SNPs) • There are more than enough (1/30kb thus >>1/cM) • Reliable genetic maps (Marshfield, Decode) • Optimized marker sets, spacing down to 5cM (Marshfield/Applied Biosystems) • Reasonably automated measurement (2 persons  40,000 checked genotypes in database per week) • Low cost per genotype (<$0.15 for consumables) • Reasonable success and error rates (>92% and <0.8%)

  5. AACTAACTAACTAACT TTGATTGATTGATTGA AACTAACT TTGATTGA Short tandem repeats Tetranucleotide repeat: Paternal allele 4 repeats Maternal allele 2 repeats

  6. AACTAACTAACTAACT TTGATTGATTGATTGA AACTAACT TTGATTGA Short tandem repeats Tetranucleotide repeat: Paternal allele 4 repeats Maternal allele 2 repeats Dinucleotide repeat: Paternal allele CACACACACACACACA GTGTGTGTGTGTGTGT 8 repeats CACACA GTGTGT Maternal allele 3 repeats And there also are tri- and pentanucleotide repeats….

  7. Principle of genotyping methods • Short tandem repeats  length differences CACACACACACACACA GTGTGTGTGTGTGTGT CACACA GTGTGT • SNPs  only sequence difference • Destruction restriction site (RFLP) • Hybridization differences (TaqMan) • One base-pair sequencing reaction- primer extension (Sequenom, Orchid) • Ligation assay (Illumina) G C A T • VNTR, insertion/deletion polymorphisms (1 bp to ~300 bp for Alu repeat)

  8. Genotyping STRs – step 1: PCR

  9. Genotyping STRs – step 1: PCR CACA GTGT 104 bp 20 + 25 + 4 + 35 + 20 = CACACACA GTGTGTGT 108 bp 20 + 25 + 8 + 35 + 20 =

  10. Genotyping STRs – step 1: PCR in practice genomic DNA + primers + Taq DNA polymerase + dNTPs (ACGT) + buffer

  11. Genotyping STRs – step 2: electophoresis Detect length differences • Agarose or polyacrylamide slab gel • DNA is negatively charged • Longer fragments migrate slower than shorter ones through polymer network. — electrode + electrode

  12. To scan the whole human genome… • 1 short tandem repeat every 10 cM • makes 400 markers per individual • Assuming 1000 individuals (preferably 1000s) • One whole genome scan = 400,000 genotypings

  13. Not like this…….

  14. Not like this……. but like this 96-well plates 384-well plates

  15. Not like this…….

  16. Not like this……. but like this

  17. Not like this…….

  18. Not like this……. but like this

  19. - TCTC AGAG start Typically 15 markers in one capillary: TGTGTG ACACAC GTGT CACA Detector CACA GTGT Laser 2.5 h A bit later + Electrophoresis using automated sequencer • 96 capillaries (no lanes) (ABI3700) • Put in machine and all goes automatically • Primers are labelled with fluorescent dye • Machine detects PCR products through a laser

  20. Through-put • A 384-well plate taking about one night • 384 samples minus 16 controls = 368 • 15 markers per sample • makes 5520 genotypes (if succes rate 100%)

  21. Tetranucleotide repeat marker (e.g. multiples of AACT)

  22. Detected length of PCR product depends on machine • Standards are used to correct this (CEPH DNA samples) • Take this into account when analysing data from different machines/labs

  23. Dinucleotide repeat marker (e.g. multiples of CA)

  24. Dinucleotide repeats give less clean pictures but in practice this is no problem as long as pattern is always the same • However, markers not in standard 10 cM screening sets often are more problematic (different stutter patterns for different samples, non-constant ratio ‘real peak’/plus-A peak) •  increased error rates?

  25. The result: allele lengths CACA GTGT 104 bp 20 + 25 + 4 + 35 + 20 = CACACACA GTGTGTGT 108 bp 20 + 25 + 8 + 35 + 20 =

  26. Renumbered data 1 3 2 2 0 0 5 6 5 2 4 3 4 0 0 8 7 7 Pedigree file in linkage format Raw marker data 1 1 1 1 2 2 2 2 2 1 2 3 4 1 2 3 4 5 0 0 1 1 0 0 1 1 1 0 0 2 2 0 0 2 2 2 1 2 2 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 102 106 104 104 0 0 111 112 111 104 110 106 110 0 0 118 114 114

  27. Genetic map of measured markers • For IBD estimation using Merlin or other software • Pedigree file • Genetic map

  28. Markers measured on chromosome 19 16 markers d19s247 d19s1034 d19s391 d19s865 d19s394 d19s588 d19s49 d19s433 d19s47 d19s420 d19s178 apoc2 d19s246 d19s180 d19s210 d19s254

  29. Genetic maps • Available from • Marshfield Center for Medical Genetics • http://research.marshfieldclinic.org/genetics/ • Decode Genetics (most accurate) • Supplemental data to Kong et al. Nat Genet 2002;31:241-7. • see F:\Bas\Genotyping&Maps\DecodeMap.xls

  30. Merlin Map File CHROMOSOME MARKER LOCATION 19 d19s247 9.84 19 d19s1034 20.75 19 d19s391 28.83 19 d19s865 32.39 19 d19s394 34.25 19 d19s588 42.28 19 d19s49 50.81 19 d19s433 51.88 19 d19s47 63.10 19 d19s420 66.30 19 d19s178 68.08 19 apoc2 69.50 19 d19s246 78.08 19 d19s180 87.66 19 d19s210 100.01 19 d19s254 100.61

More Related