1. Linkage analysis & Homozygosity mapping
Practical Session
Linkage analysis & Homozygosity mapping
Wouter Steyaert 1

2. Practical session: content of the presentation
Introduction
Superlink SNP Online (GUI)
Merlin (command line)
HomozygosityMapper

3. Interpretation of results
Introduction
Linkage: mapping the location of disease-causing loci by identifying genetic markers that are co-inherited with a phenotype of interest
Types of information
Superlink
Merlin
Markers
SNP file
DAT file &
MAP file
Pedigree
PED file
PED file &
MODEL file
Algorithm + computer
Interpretation of results

4. + Powerful + User-friendly (GUI) +/- External cloud
Superlink SNP Online
+ Powerful
+ User-friendly (GUI)
+/- External cloud 4

5. Superlink SNP Online: SNP file
HumanCytoSNP-12 BeadChip on HiScan instrument (Illumina)
genotypes/patient
Cannot directly be used in Superlink/Merlin
conversion

6. Superlink SNP Online: SNP file
Superlink SNP Online SNP file = 1 data file for whole pedigree
Conversion via Perl/Python/…

7. Superlink SNP Online: PED file
Simplified PED-format (notepad)
Column 1: id
Column 2: id of the father
Column 3: id of the mother
Column 4: sex (1=Male, 2=Female)
Column 5: status (0=Unknown, 1=unaffected, 2=affected)

8. Superlink SNP Online: wizard
Choose input format 8

9. Superlink SNP Online: wizard
Upload data 9

10. Superlink SNP Online: wizard
Set parameters
e.g. homozygous
for whole family
If not fully penetrant power will drop! 10

11. Superlink SNP Online: HMM_MultiPoint
Powerful, but computationally expensive
only possible for small pedigrees
Max LOD = 2 because pedigree is small ( 6 typed individuals) ≈
102 times more likely that the marker is linked with the disease causing mutation than it is not linked with the mutation 11

12. Superlink SNP Online: Exact multi-point analysis
Possible for all pedigrees
Set parameters 12

13. Superlink SNP Online: Exact multi-point analysis
Fetch genes and discover (e.g. via BioMart)
Max LOD = 2,8

14. Superlink SNP Online: Exact multi-point analysisB A B A B A B A B A B A B A B A
All affected individuals should have 1 haplotype in common. The causal haplotype? Yes, with a certain probability.
BUT…

15. Linkage analysis: difficulties
Uncertainty about the clinical status of some individuals
 exclude those individuals (but power will drop)
Uncertain relationships (software can correct for this)
Incomplete penetrance
Phenocopies
Statistically significant linkage (LOD > 3,2)
≠ usefulness of the analysis
Prioritize regions
Exclude regions (f.e. known disease causing genes)
Often followed by exome analysis

16. + Available for Linux/Win/Mac + Powerful +/- Command line
Merlin
+ Available for Linux/Win/Mac
+ Powerful
+/- Command line

17. Merlin: PED file
= simple PED file
(Superlink)
+ extra columns
1) Family ID
7) Genotype first marker
8) Genotype second marker …
(purple block is 1 individual)
Conversion from RAW Illumina data via Perl/Python/…

18. Merlin: DAT file
= list of markers used in the analysis
= selection of all markers
f.e. randomly select 1 SNP/ bp
from to SNPs
+ filter, f.e. non informative SNPs
(Superlink does this automatically)

19. Merlin: MAP file
The same markers as in DAT files, but accompanied with
Chromosome
Genetic position (in centiMorgans)
Rutgers Maps
Approximation: 1 cM = 1 million base pairs
= distance between chromosome positions for which the expected average number of intervening chromosomal crossovers in a single generation is 1%.
genetic distance ≠
physical distance

20. Merlin: MODEL file
Tab delimited file containing 4 columns:
Affection status label
Disease allele frequency
Probability of being affected for individuals with 0, 1 and 2 copies of the disease allele
Label for the analysis model
Necessary for parametric analysis

21. Merlin: pedstats
prompt> pedstats -d merlin.chr1.dat -p merlin.chr1.ped
verify that input files are being interpreted

22. Merlin: merlin + pedwipe
identify problem genotypes
prompt> merlin -d error.dat -p error.ped -m error.map --error
prompt> pedwipe -d error.dat -p error.ped
prompt> merlin -d wiped.dat -p wiped.ped -m error.map --npl

23. Merlin: merlin
non-parametric
prompt> merlin -d asp.dat -p asp.ped -m asp.map --pairs --npl --pdf

24. Merlin: merlin
parametric
prompt> merlin -d parametric.dat -p parametric.ped -m parametric.map --model parametric.model --step 3 --pdf

25. Homozygosity mapping
Homozygous streches
Present in group 1 (cases)
Absent in group 2 (controls)
consanguinity
Heterozygous deletion

26. Homozygositymapper: wizard
DBSNP 14 12 10 11 9 8 7 15 17 13 16 rs AB BB rs AA rs
Slightly different format

27. Wizzard
all affected are homozygous!
to avoid false positive

28. Results

29. Homozygosity mapping
SNPs
SNPs
cases
controls
Chr. 22
Chr. 5