Building genetic maps can be challenging and sometimes quite stressful, especially, when dealing with thousands or even millions of markers. In this post, I am hoping to help anyone who would like to get started to build a decent genetic map in an open software Lep-MAP3 , and finally, evaluating the accuarcy of the map and plotting it.
The steps invloved in the genetic mapping process in Lep-MAP3 are shown in the flow chart below.
Step 1.1. Installation and File Preparation
Important - Correctly install the Lep-MAP3 software on your computer, and please make sure its the latest version. There are two files that are needed as an input
Step 1.2. Parent Call
The parental genotypes are called using the
ParentCall2 module, using the below command:
$ java -cp /path/Lep-MAP3/bin ParentCall2 data = pedigree.txt vcfFile = File.vcf > p.call
Step 1.3. Filtering
One may use the
Filtering2 module to remove
non-informative markers (Markers that are monomorphic or homozygous in both parents), and similarly, to remove
distorted markers (markers segregating in a non-Mendelian fashion) using the below command line:
$ java -cp /path/Lep-MAP3/bin Filtering2 data=p.call removeNonInformative=1 dataTolerance=0.001 > p_fil.call
Note: Use: the parameter
removeNonInformative to remove markers that are homozygous/monomorphic, and
dataTolerance to remove distorted markers at given p-value threshold.
Step 1.4. Separate Chromosomes
In this step,
SeparateChromosomes2 module is used to categorize markers into linkage groups (LGs) using the below command:
$ java -cp /path/Lep-MAP3/bin SeparateChromosomes2 data=p_fil.call lodLimit=10 > map.txt
Note: Use the parameter
lodLimit to split the linkage groups.
Step 1.5. Order Markers
In this step, markers separated into their corresponding linkage groups are ordered using
OrderMarkers2 module using the below command:
$ java -cp /path/Lep-MAP3/bin OrderMarkers2 data=p_fil.call map=map.txt > order.txt
One may use the parameter
sexAveraged to calculate sex-averaged map distances ( by default male and female genetic maps are curated separately), also
numMergeIterations paramteres are used to adjust number of iterations (by deafault its is 6 iterations per linkage group).
2.0 Checking the accuracy of the marker order
If the physical positons of the markers in the genetic map curation are known, then, one may use that information to evaluate the quality of marker order, especially markers that inflate the chromosome length, by making a correlation plot of the genetic and physical positions of the markers by individual chromosomes or linkage groups. Note: It is quite common to see that the marker orders are flipped. There is nothing to panic about, one may fix it by manually sorting it.
3.0 Converting phased output data from OrderMarkers2 to genotypes
The phased data from
OrderMarkers2 step can be converted to fully informative “genotype” data by using
map2gentypes.awk script and command below:
Download the map2gentypes.awk script here.
Next, run the map2genotypes.awk script by following the command shown below.
$ awk -vfullData=1 -f map2genotypes.awk order.txt > genotypes.txt
Snippet of the
One may convert the genotypes in 1 1 => A, 2 2 => B, 1 2 or 2 1 => H format (See below figure) in MS Excel using find/Replace function, which can be then loaded in
R/Qtl for QTL mapping.
4.0 Validate the genetic map by conducting QTL analysis
5.0 Graphical presentation of linkage maps in Mapchart
Software Mapchart can be downloaded from below link. https://www.wur.nl/en/show/Mapchart.htm
Thank you for reading this tutorial. I really hope these steps will get you started in genetic map construction in Lep-MAP3. The key is to PRACTISE. . If you have any comments or suggestions, please let comment below or send me an email.
Happy mapping !
Rastas, Pasi. “Lep-MAP3: robust linkage mapping even for low-coverage whole genome sequencing data.” Bioinformatics 33.23 (2017): 3726-3732.