Add color tag to bam alignedReads using pysam

Question

[pyKey]

Hello everyone,

I have some STAR_aligned bam file. I want to add color tags to some reads in the file using pysam, in order to track them better in IGV.

Here is the code part which adds the tag:

red = '255,0,0'

r = pysam.AlignedRead()

r.tags += [('YC', red)]

This updates the file with new tag, which however does not work properly in IGV. I think some casting is missing here which I cannot think of.

Has anyone done this before? Thank for your help :)

[Pierre Lindenbaum]

can you see this YC tag in your bam using samtools view ?

[pyKey]

Yes, and it looks like a decent one:

HISEQ:280:C9J9KANXX:2:2101:7621:44090   16  chr10   31332804    255 50M *   0   0   GAGCAGGCAGATTGACCAGCAGCAGCCCAGGCGGGACCTAGCACTGCAGC  FFGGFGGGEFDEFFGGGGGFGGFGF0GGGFFGGGGGGF;GGGGGDCBBB@  XC:Z:CCGGAC GE:Z:ENSMUSG00000000296 XF:Z:CODING PG:Z:STAR   RG:A:A  NH:i:1  NM:i:0  XM:Z:GAAGTTATGG UQ:i:0  AS:i:49 GS:A:-  YC:Z:255,0,0

But in IGV I get this error once I try hover over these reads (and of course no view):

"Error encountered querying alignments: java.lang.ClassCastException: java.lang.Character cannot be cast to java.lang.String"

[pyKey]

Hi Pierre, you are right one tag is not of correct type. That's curious! Please take a look at these two queries of the same read, before and after adding the color tag:

HISEQ:280:C9J9KANXX:1:1210:16223:39511  16  chr10   3176253 255 39M7965N7M4S    *   0   0   TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAACCTTATGC  GGGGDGGGDGGGGGGGGGGGGGGGGGGGGGGDDGGGCB=00000>CBB@A  XC:Z:CCGGAC XF:Z:CODING PG:Z:STAR   RG:Z:A  NH:i:1  NM:i:0  XM:Z:AGAAAGGTGG UQ:i:0  AS:i:45

after:

HISEQ:280:C9J9KANXX:1:1210:16223:39511  16  chr10   3176253 255 39M7965N7M4S    *   0   0   TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAACCTTATGC  GGGGDGGGDGGGGGGGGGGGGGGGGGGGGGGDDGGGCB=00000>CBB@A  XC:Z:CCGGAC XF:Z:CODING PG:Z:STAR   RG:A:A  NH:i:1  NM:i:0  XM:Z:AGAAAGGTGG UQ:i:0  AS:i:45 YC:Z:255,0,0

I did not make any changes to the RG tag, but somehow its type has changed. Any theories? Tag parsing maybe??

[Pierre Lindenbaum]

yes, you should change your read group to a two-letter code.

Answer 1

[Pierre Lindenbaum]

HISEQ:280:C9J9KANXX:2:2101:7621:44090 16 chr10 31332804 255 50M * 0 0 GAGCAGGCAGATTGACCAGCAGCAGCCCAGGCGGGACCTAGCACTGCAGC FFGGFGGGEFDEFFGGGGGFGGFGF0GGGFFGGGGGGF;GGGGGDCBBB@ XC:Z:CCGGAC GE:Z:ENSMUSG00000000296 XF:Z:CODING PG:Z:STAR RG:A:A NH:i:1 NM:i:0 XM:Z:GAAGTTATGG UQ:i:0 AS:i:49 GS:A:- YC:Z:255,0,0

the problem comes from your RG flag. The specification says it must be a String RG:Z:A but you have a character RG:A:A

I've created a tool to inject a YC tag using a javascript expression: http://lindenb.github.io/jvarkit/SamColorTag.html

I've just tested it with IGV 2.2.4 , it works fine: