STAR can not detect this chimeric read

Question

[Nicolas Rosewick]

Hi,

I try to detect chimeric fusions between a virus (integrated into the host genome) and the host genome. I aligned the reads (paired-end 2x76 stranded) on a hybrid genome (host+virus) where the virus genome is considered as an additional chromosome. Here's my command :

$STAR --genomeDir $stargenomeDir --outFilterScoreMinOverLread 0.3 --outFilterMatchNminOverLread 0.3 -seedSearchStartLmax 10 --outFilterMultimapNmax 10 --outFilterMismatchNmax 10 --chimSegmentMin 10 --outFilterMatchNmin 10 --chimJunctionOverhangMin 10 --readFilesIn $r1 $r2 --runThreadN $threads --outStd SAM --readFilesCommand zcat

version : STAR_2.3.1u_r375

So I expect STAR to report fusion reads with minimum 10 bases aligning either of the host or virus ; and the rest on the virus or the host respectivelly. As :

 # : host genome
 @ : virus genome
 = : read
 - : splicing

#######################################@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
          ==========-----------------------------------=========================
            min 10bp

But when I check on IGV on the extremity of virus genome I observe some reads containing soft-clipping longer than 10bp (in this cases they are 13bp long). When I align these soft-clipped bases on the host genome using blast I found a position on the host genome where I indeed found traces of a fusion transcript which continues (I can clearly see reads that aligned after the fusion breakpoint representing the fusion transcript). But STAR do not report this fusion. Am I doing something wrong ?

Here's the SAM lines for a pair of reads containing the soft-clipping of interest :

NS500186:93:HYFJVBGXX:3:13402:19874:18099   163 chrVirus    1   255 16S59M  =   65  128 CACATGTTTAGGTTTGTGACAATGACCATGAGCCCCAAATATCCCCCGGGGGCTTAGAGCCTCCCAGTGAAAAAC AAAAAEEEEEEEEEEEEEEEEEEEAEEEEEAEEEEEEEEEEEEEEE/EEEEEEEEEEEEAEEE<EEEEEEEEEEE NH:i:1  HI:i:1  AS:i:117    nM:i:2
NS500186:93:HYFJVBGXX:3:13402:19874:18099   83  chrVirus    65  255 64M12S  =   1   -128    CGCGAAACAGAAGTCTGAAAAGGTCAGGGCCCAGACCAAGGCTCTGACGTCTCCCCCCGGAGGGACAGCTCAGCAC    AAEEEEEEEEEEEEEAEEEEEEE/EEEEEEEAEEEAEEEEE/EEEEEEEAEEEEEEEEEEEEEEEEEAEAEAAAAA    NH:i:1  HI:i:1  AS:i:117    nM:i:2

I put two figures explaining my cases

Alignment on the virus :

Alignment on the host :

Thanks

[Nicolas Rosewick]

I think I found the issue. The soft-clipped sequence appears to be present at multiple position in the host genome. I suppose STAR do not report multi-mapping fusions. I'll dig a little bit more to be sure.

[GenoMax]

@NicoBxl: Both of the links you included for the images are not loading (I tested them). Can you post new versions and update your post?

[Nicolas Rosewick]

Should work. The links are ok. I also put the SAM lines for a pair of reads.

[GenoMax]

Perhaps the links are working from your part of the world but they still don't work for me. Referring to this link for example.

[Nicolas Rosewick]

I reupload the picture and change the post. You see them now ?

[GenoMax]

I still can't (even if I use the URL) but don't worry. It may be local firewall specific. Other's able to see the images?

[WouterDeCoster]

I see images. Even when sober.

[Devon Ryan]

I see them even before coffee :P

@NicoBxl: You might post this to the STAR email list/google group. Alex is usually pretty good about replying (given how many options STAR has, I expect he's the only one that can point to the right one to tweak).

[Nicolas Rosewick]

Yes I will do that. I will post his answer here.

[Asaf]

If you would like to try another tool I wrote RILseq which was built for chimera detection in bacteria but I don't see why it wouldn't work here. It overcomes multiple mapping reads and will do some statistical analysis to detect over-represented chimera. See https://pypi.python.org/pypi/RILseq

[Ron]

Did you try STAR-fusion?