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Old 04-14-2013, 07:59 PM   #1
angerusso
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Exclamation tophat2: missed 5/27 breast cancer cell line fusions

I missed 2 BT474 and SKBR3 fusions using tophat2. I think I was supposed to find 25/27 of these fusions according to tophat paper. Can anyone help me with why could this be? See below my parameters:

BT474 CPNE1-PI3
BT474 LAMP1-MCF2L
SKBR3 CCDC85C-SETD3
SKBR3 CSE1L-ENSG00000236127
SKBR3 WDR67-ZNF704

> tophat2 -o $file -r 24 -p $np --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --segment-mismatches 2 --segment-length 25 --fusion-ignore-chromosomes chrM $index $read1 $read2

> tophat-fusion-post -p $np --num-fusion-reads 1 --num-fusion-pairs 1 --num-fusion-both 2 $index
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Old 04-15-2013, 02:33 AM   #2
ndaniel
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Hello,


here one may found all the gene fusions known to exist and validated in SKBR3 and BT474 cell lines.

- S. Kangaspeska, S. Hultsch, H. Edgren, D. Nicorici, A. Murumägi, O.P. Kallioniemi, Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms, PLOS One, Oct. 2012. http://dx.plos.org/10.1371/journal.pone.0048745
- H. Edgren, A. Murumagi, S. Kangaspeska, D. Nicorici, V. Hongisto, K. Kleivi, I.H. Rye, S. Nyberg, M. Wolf, A.L. Borresen-Dale, O.P. Kallioniemi, Identification of fusion genes in breast cancer by paired-end RNA-sequencing, Genome Biology, Vol. 12, Jan. 2011. http://genomebiology.com/2011/12/1/R6

For finding all these fusion genes FusionCatcher <http://code.google.com/p/fusioncatcher/> has been used!

Here are the fusions which FusionCatcher finds in SKBR3
http://code.google.com/p/fusioncatch...i/fusionsSKBR3

Here are the fusions which FusionCatcher finds in BT474
http://code.google.com/p/fusioncatch...i/fusionsBT474

Have you tried do find gene fusion using FusionCatcher?

Daniel

Quote:
Originally Posted by angerusso View Post
I missed 2 BT474 and SKBR3 fusions using tophat2. I think I was supposed to find 25/27 of these fusions according to tophat paper. Can anyone help me with why could this be? See below my parameters:

BT474 CPNE1-PI3
BT474 LAMP1-MCF2L
SKBR3 CCDC85C-SETD3
SKBR3 CSE1L-ENSG00000236127
SKBR3 WDR67-ZNF704

> tophat2 -o $file -r 24 -p $np --fusion-search --keep-fasta-order --bowtie1 --no-coverage-search --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --segment-mismatches 2 --segment-length 25 --fusion-ignore-chromosomes chrM $index $read1 $read2

> tophat-fusion-post -p $np --num-fusion-reads 1 --num-fusion-pairs 1 --num-fusion-both 2 $index
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Old 04-15-2013, 08:03 AM   #3
angerusso
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Default Missing 4 fusions using tophat2

Hi Daniel,

It seems you mis understood my question. the question was how to modify tophat2 or tophat-fusion-post parameters to find the 4 missing fusions which were found using previous versions of tophat as reported in Kim and Salzberg publication?

Thanks, Angel
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Old 04-15-2013, 10:49 AM   #4
ndaniel
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I am sorry. I was confused about your question "Can anyone help me with why could this be?".

Also I am confused to which 27 fusions are you referring to? The paper which you are referring to (that is Kim et al. "Tophat-fusion: An algorithm for discovery of novel fusion genes", Genome Biology 2011) has published over 1,395 fusion genes only in SKBR3 cell line (see: http://genomebiology.com/content/sup...2-8-r72-s9.txt )!!!!

Are you using exactly the same version of:
1) Tophat-fusion, and
2) genome, and
3) annotation
like in the paper which you mentioned?
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Old 04-15-2013, 12:02 PM   #5
angerusso
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Question

Hi Daniel,

I am referring to the 27 fusions that are mentioned to be known and/or validated by RT-PCR as mentioned in the paper.

I didn't use the same version of software as in the paper. I used the following and wondering why I am missing the 4 fusions out of 27 that I am supposed to get using the exact same parameters are mentioned in the paper:

tophat/2.0.8
samtools/0.1.19
bowtie1 and bowtie/0.12.9
blast/2.2.27+

Thanks.
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Old 04-16-2013, 02:14 AM   #6
ndaniel
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You wrote:
================================
I am referring to the 27 fusions that are mentioned to be known and/or validated by RT-PCR as mentioned in the paper.
================================

If before I was confused now I am completely lost. To which fusion 27 genes are you referring? What Table? A table number or figure number would help greatly here!

I found the word "PCR" only once in the whole article (Kim et al, Tophat-fusion) in this sentence "Next, we ran all of FusionSeq’s filters except two PCR filter and annotation consistency filter) that would otherwise eliminate two of the true fusions" which has nothing to do with the validation of gene fusions. Also the authors of the same article have not done any validation using RT-PCR on their newly found fusion genes.

You wrote:
================================
I didn't use the same version of software as in the paper. I used the following and wondering why I am missing the 4 fusions out of 27 that I am supposed to get using the exact same parameters are mentioned in the paper.
================================


Not using the exact version of annotation database might explain why you do not find all the gene fusions from the article. From my experience with FusionCatcher and SOAPfuse some gene fusions can be found only using a very specific version of annotation database (like Ensembl in the case or the article).

You missed the fusion CSE1L-ENSG00000236127 in SKBR3 cells. In the same article is written about this fusion "one known fusion, CSE1L-ENSG00000236127, was not considered because ENSG00000236127 has been removed from the recent Ensembl database". From what I understand you are using a newer version of Ensembl database and this explains why you cannot find the CSE1L-ENSG00000236127.
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