Use the latest version of Circos and read Circos best practices—these list recent important changes and identify sources of common problems.
If you are having trouble, post your issue to the Circos Google Group and include all files and detailed error logs. Please do not email me directly unless it is urgent—you are much more likely to receive a timely reply from the group.
Don't know what question to ask? Read Points of View: Visualizing Biological Data by Bang Wong, myself and invited authors from the Points of View series.
The default order of ideogram display is the order of appearance in the karyotype definition file. Display of individual ideograms can be toggled using the chromosome field (see previous tutorial), but this field does not influence the order of display. For this, the chromosomes_order field is used.
The simplest way to specify a new order is to exhaustively list the new order of ideograms. For example,
chromosomes = hs1;hs2;hs3;hs4;hs5;hs6;hs7;hs8 chromosomes_order = hs2,hs3,hs1,hs5,hs4,hs8,hs7,hs6
The new order is 2 3 1 5 4 8 7 6.
Note that the delimiter for the order field is a comma (,), whereas a semicolon (;) is used for the list of ideograms to draw. The reason for this will be explained later.
If you have a small number of ideograms, or your custom order is always fixed then a full ordered list is practical. In cases where you want to adjust order by specifying the position of one ideogram relative to another, a more complex syntax for chromosomes_order is used.
You can specify ideogram order for a subset of ideograms by specifying local order. For example, to specify that hs4 should follow hs5,
chromosomes_order = hs5,hs4
In this case, the order is 1 2 3 6 5 4 7 8, which you may find surprising (the fact that 6 comes before 5). When Circos parses a relative order, such as hs5,hs4, it looks at the first named ideogram, in this case hs5, and uses that as an anchor for ordering remaining ideograms in the order list. Since hs4 is specified after hs5, Circos places hs4 immediately after hs5, but does not move the original location of hs5. Since hs4 has been moved, the next ideogram (hs6) takes its place.
In order to achieve order 1 2 3 5 4 6 7 8, you need to anchor on hs3.
chromosomes_order = hs3,hs5,hs4
Remember, the first named entry in the order list will be the anchor and this should correspond to the ideogram that you don't want to move. The remaining ideograms in the list will be relocated relative to the anchor.
Instead of the chromosome ID, you can enter "-" in an order list to indicate the next available ideogram. Here, the sense of "next" is relative to default order.
The purpose of the wildcard "-" entry is to allow for additional flexibility on ordering the ideograms. For example, to specify that hs2 should be the next neighbour after hs3,
chromosomes_order = hs3,-,hs2
This order list would produce the order 4 5 3 1 2 6 7 8. Again, the result may be a little surprising. What has happened is that the order request used hs3 as the anchor ideogram. Thus, the position of hs3 in the final order is not altered (it comes 3rd). The next ideogram is "-", which is a wildcard that indicates the next available ideogram, or hs1 since this is the first ideogram that has not been specifically mentioned in the order list. Thus the local order in the vicinity of hs3 is hs3, hs1, hs2. Since hs3 is 3rd, the first two slots are replaced by the first available ideograms, hs4 and hs5.
If you want two ideograms to separate hs3 and hs2, then
chromosomes_order = hs3,-,-,hs2
would produce the order 5 6 3 1 4 2 7 8, anchoring on hs3. If however you enter
chromosomes_order = hs2,-,-,hs3
then the anchor is hs2 and the order is 5 2 1 4 3 6 7 8.
You can intersperse wildcards with specific ideograms. For example
chromosomes_order = hs1,-,hs2,-,hs3
would produce 1 4 2 5 3 6 7 8. The wildcards are filled by the first available ideograms that were not specifically ordered - these are hs4 and hs5.
You can place a wildcard as the first entry, to force an ideogram to appear before your anchor. For example,
chromosomes_order = -,hs1,-,hs2,-,hs3
yields 4 1 5 2 6 3 7 8. In this case, the rule that the anchor's (here hs1) position is unchanged is overridden by the fact that you've requested an ideogram to appear in front of the anchor. Effectively, you have forced the insertion of the first available ideogram before hs1.
In addition to the "-" wildcard in the order list, you can use two additional metacharacters: the ^ and $ anchors. ^ indicates the beginning of the circular map and $ indicates the end. Thus, to have hs5 appear as the first ideogram, it is enough to say
chromosomes_order = ^,hs5
and the order will be 5 1 2 3 4 6 7 8. If you would like hs5 to appear last,
chromosomes_order = hs5,$
and now the order will be 1 2 3 4 6 7 8 5.
Multiple order groups can be specified by using '|' as a delimiter. Each group affects order local to its anchor (the first specified ideogram within the group).
Consider the following order string,
chromosomes_order = hs3,hs2,|,hs8,hs1
and note the '|' that separates the two groups hs3,hs2 and hs8,hs1. The anchor for the first group is hs3 and hs2 is ordered relative to it. The anchor for the second group is hs8 and hs1 is ordered relative to it. The final order is 4 5 3 2 6 7 8 1.
When using multiple groups, wildcards can add additional flexibility. Consider the following order string
chromosomes_order = hs3,-,hs2,|,-,hs8,hs1
Each group is evaluated independently, but in order of appearance. Thus, the first wildcard is filled by hs4, which is the first ideogram that hasn't been explicitly placed. The wildcard in the second group is the next available ideogram, and that is hs5 (since hs4 has already been used by the previous wildcard). The final order is 6 7 3 4 2 5 8 1.
The strength of specifying multiple groups is realized when you have a lot of ideograms to draw, and want to specify order for several local sets of ideograms without having to write down the order for all ideograms.
Finally, you can combine wildcards, multiple groups and anchors in one string. This can quickly yield a complex order string.
chromosomes_order = ^,-,hs5,hs2,|,hs4,hs3,|,hs7,$
The final order is 1 5 2 4 3 6 8 7.
When constructing a string with multiple groups, wildcards and anchors, each individual rule must be fulfilled, or Circos exists with an error. It is your responsibility to write down rules that describe a consistent global order. For example, if the number of wildcards and ideograms in your order string is larger than the number of ideograms you are drawing, Circos will produce an error. For example, this string requires at least 8 ideograms
chromosomes_order = ^,-,hs5,hs2,|,hs4,-,-,hs3,|,hs7,$
and works with our example to produce an order 1 5 2 4 6 8 3 7. However, if one more wildcard is added
chromosomes_order = ^,-,hs5,hs2,|,hs4,-,-,-,hs3,|,hs7,$
we run out of ideograms to place and get an error
fatal error - chromosomes_order string cannot be processed because group hs7 cannot be placed in the display. This may be due to more tags in the chromosomes_order field than ideograms.