To group large numbers of markers spanning a genome, we need to develop a slightly different notion of linkage. For the mouse genome, a LOD score of 3.0 represents about a 1 in 20 significance level, meaning that in the roughly 50,000 pairwise comparisons, we will falsely declare over 2,000 linkages! Because of transitivity, as we discussed earlier, many chromosomes may become linked into any one linkage group.
The solution is not necessarily to blindly raise the LOD threshold: to raise it high enough will exclude a large number of true linkages. For this reason we will not use MAPMAKER's "group" command, but instead we use the "assign" command. Here's how it works:
In the initialization file, certain markers have been pre-assigned to chromosomes: this will determine which linkage groups belong on which chromosomes. (These are the so called "anchor" loci). To assign the rest of the markers to chromosomes we:
The algorithm is in fact slightly more complex than this, in order to deal with conflicting data well (cases where a marker shows linkage to more than one chromosome). See the description in the reference section for details.
To assign all markers in the data set to chromosomes, we first set MAPMAKER's sequence. As we do not wish to type in the number or name of each locus, we will use the special abbreviation "all". Next we simply type "assign".
As you see, MAPMAKER's "assign" command produces quite a bit of output, indicating the strength of assignment for each of the markers in the data set. Thankfully, MAPMAKER provides efficient ways of viewing these results.
55> sequence all sequence #21= all56> assign L016 - anchor locus on chrom12...cannot re-assign L018 - anchor locus on chrom2...cannot re-assign L019 - anchor locus on chrom9...cannot re-assign L020 - anchor locus on chrom1...cannot re-assign .
<< More Output Follows .
L036 - assigned to chrom17 at LOD 10.4 L046 - assigned to chrom1 at LOD 3.6 L010 - assigned to chrom15 at LOD 18.1 D004 - assigned to chrom9 at LOD 13.1 L007 - assigned to chrom16 at LOD 21.1 D016 - assigned to chrom17 at LOD 9.9 D021 - assigned to chrom17 at LOD 21.1 D017 - assigned to chrom15 at LOD 3.1 .
<< More Output Follows .
M251 - unassigned A066 - unassigned
One way to summarize the chromosome assignments is to use MAPMAKER's "list chromosomes" command. This simply prints a summary of the total number of markers assigned to each chromosome (in the first column) as well as other information for analyses we have not yet performed.
As you can see in the "Total" line, not all markers were assigned (that is, only 305 of the 308 were assigned). In fact, if you review the "assign" command's output, you will see that markers M228, M251 and A066 remain unassigned. Your computer may or may not provide a convenient way to scroll backwards through the output (PC's in particular lack this ability). If this is the case, you may try using the "review output" command, described in the reference section.
As a different way to review a summary of these results, we may use the "list status" command. To see the information for only the three unassigned markers, we first change the sequence, using another convenient "sequence" abbreviation: "unassigned".
As you can see, conflicting data exist for locus M228 -- it shows evidence of linkage to two different chromosomes. This may indicate a possible problem in the raw data, or it may be the result of statistical fluctuation: After performing almost 50,000 pairwise tests, it is not unlikely to see a spurious linkage at LOD 3.8. The other two markers, M251 and A066, showed no conclusive evidence of linkage.
To provide more information, we can also easily generate a list of all loci linked to the three unassigned markers. For this, we use MAPMAKER's "links" command. At a LOD threshold of 3.0 (the default) we see the basis to the conflicting linkage data for M228 -- It shows evidence of linkage to markers on both chromosomes 3 and 14 (although certainly the evidence for linkage to chromosome 14 is much stronger than the evidence of linkage to chromosome 3 (highest LOD of 11.75 vs. 3.58). This suggests that the chromosome 3 linkage may in fact be spurious -- if we check the data and come to believe the chromosome 14 assignment, we could then simply assign M228 to chromosome 14 using MAPMAKER's "attach" command.
By contrast, marker A066 appears truly unlinked, although this could in principle be due to true recombinational distance or massive data errors. Marker M251 shows weak evidence of linkage to chromosome 13 -- it may in fact be quite distant, although this too would be a good marker to double check in the lab.
57> list chromosomes Chromosome: #Anchors #Total chrom1 2 18 chrom2 2 30 chrom3 2 21 chrom4 2 19 chrom5 2 12 chrom6 2 19 chrom7 2 20 chrom8 2 14 chrom9 2 22 chrom10 2 15 chrom11 2 15 chrom12 2 12 chrom13 2 11 chrom14 2 9 chrom15 2 18 chrom16 2 7 chrom17 2 22 chrom18 2 16 chrom19 2 5 Total: 38 30558> sequence unassigned sequence #22= unassigned
59> list status Num Name Assignment Chrom LOD 181 M228 conflict! - - 196 M251 unlinked - - 274 A066 unlinked - -
60> links Linked Markers in Data Set at min LOD 3.00, max Distance 50.0 Marker-1 Marker-2 Theta LOD cM M228 L038 0.18 3.58 23.00 (chrom3) M228 T010 0.16 5.55 19.13 (chrom14) M228 M032 0.05 11.75 5.28 (chrom14) M228 M214 0.08 9.70 8.28 (chrom14) M228 A024 0.13 6.51 15.22 (chrom14) M228 A034 0.20 3.37 24.73 (chrom3) M228 A060 0.17 3.81 21.27 (chrom3) M228 A103 0.17 5.13 21.20 (chrom14) M228 A119 0.14 6.43 16.01 (chrom14) M251 no linked markers A066 no linked markers