Executables required: quiklink, makeped, unknown, mlink, linkmap, table (optional), gnuplot (optional), easigraf (optional), ACE/gr (optional), Excel (optional), lrp (optional).
In previous exercises we have modified the example qdb database to incorporate genotypes for a third marker locus and have produced a LINKAGE pedigree file called autdom3.ped. This pedigree file contains data for an affection locus and three marker loci. The earlier version of the locus data file, autdom2.par, contains information about the affection locus and first two marker loci. We have provided a modified version of this file which includes information concerning the third marker.
Use the text editor to examine autdom3.par. It should appear as follows:
4 0 0 5 << no loci, risk locus, sexlinked(if 1) 0 0.0 0.0 0 << mut locus, mut rate, haplotype freq(if 1) 1 2 3 4 << order of loci 1 2 # DIS1 0.9995 0.0005 << gene freqs 1 << number of liability classes 0.0 1.0 1.0 3 4 # MAR1 0.14 0.32 0.21 0.33 << gene freqs 3 3 # MAR2 0.4 0.4 0.2 3 3 # MAR3 0.3 0.4 0.3 0 0 0.0 0.1 0.1 1 0.05 0.4
In order to accommodate information for the third marker, the following modifications have been made:
Before being able to use quiklink to set up linkage analyses with the new files, pointers and probands must be added to the pedigree file using makeped, so at the operating system prompt enter:
makeped autdom3.ped autdom3.ppd n
Now that the appropriate pedigree and locus data files have been prepared it is possible to set up some linkage analyses incorporating the third marker. To set up a simple two-point mlink analysis between the disease locus and the third marker, at the system prompt enter:
quiklink autdom3.ppd autdom3.par
Then provide the input:
d1m3 dis1 mar3
This will cause quiklink to write the files d1m3.ppd, d1m3.par and d1m3.bat (or under Unix d1m3.sh).
Suppose that the third marker lies between the first two markers, at a recombination fraction of 4% with the first one and 6% with the second one. Then we might want to set up a multipoint linkmap analysis with the disease locus tested against all three marker loci. In order to do this, when quiklink requests the filename and loci to be used in the analysis input:
d1m1m3m2 dis1 mar1 mar3 mar2
The first locus will be the test locus which will be moved along the map defined by the other three, and the other three must be in the correct order. Between the three markers there are two intervals, and quiklink will therefore request that you enter two recombination fractions. These will be the recombination fractions between MAR1 and MAR3, and between MAR3 and MAR2, so input:
0.04 0.06
You should see that quiklink writes the pedigree, parameter and command files for the multipoint analysis. Exit quiklink by pressing Enter again. You should execute the command files in order to run the analyses.
Although we have set up a couple of analyses for MAR3 interactively, in fact it would make more sense to produce an input file for quiklink which specified the parameters for the desired analyses. Suppose that after we had gone through the process above we discovered that we had entered one of the genotypes for MAR3 incorrectly. Then we would need to correct the database, re-export the .ped file, run makeped again, run quiklink and then type in the specifications for the analyses all over again. We might have forgotten what the recombination fractions between the markers were, or we might mistype these recombination fractions without noticing. For these kinds of reasons it is generally preferable to construct an input file which will set up all the desired analyses automatically. Using a text editor, modify alltest.inp to include additional instructions to set up analyses with the third marker. The additional analyses required are: a two-point analysis between the disease and the third marker; two three-point analyses taking the disease against two consecutive markers at a time; a four-point analysis of the disease against all three markers. In addition, it would be sensible to check that the markers seem to have about the expected recombination fraction with each other - doing this can throw up errors in genotyping - so you should set up two-point analyses between all possible pairs of the three markers.
When you have modified alltest.inp to include all the desired analyses save it to disk with the new name alltest3.inp. The new version of alltest3.inp might appear as follows:
d1m1 dis1 mar1 d1m2 dis1 mar2 d1m3 dis1 mar3 d1m1m3 dis1 mar1 mar3 0.04 d1m3m2 dis1 mar3 mar2 0.06 d1m1m3m2 dis1 mar1 mar3 mar2 0.04 0.06 m1m2 mar1 mar2 m1m3 mar1 mar3 m2m3 mar2 mar3It does not matter if we regenerate pedigree, parameter and command files for the analyses we have already completed. In order to set up all the desired tests automatically, at the system prompt enter:
quiklink autdom3.ppd autdom3.par <alltest3.inp
If there are any errors, correct the input file, save it to disk and then run quiklink again.
At the operating system prompt, run the command files for all the analyses which you have not yet carried out. Then use table to examine the results. Remember that the log file results which are read by table will be stored in files with extension .res. Also remember that with the multipoint analyses you can produce graph files for gnuplot, easigraf or ACE/gr using table by appending the -p, -g or -x switch, or you can use Excel to view a graph from the .tab file.
Examine the results and think how you might interpret them. Are the results of the two-point analyses between the markers plausible? What are the two-point results between the disease and the new marker? What multipoint lod score is obtained from the analyses incorporating the new marker? What is the most likely position for the disease locus?
This section goes through the steps necessary to set up additional two-point and multipoint linkage analyses with a new marker using quiklink.
Exercises in genetic linkage analysis
All material copyright (C) Dave Curtis 1996-2006
david.curtis@qmul.ac.uk