tophat with multiple fastq files

[dariober]

Hi All,

I'm probably missing something really obvious here which I can't figure out...

The problem: It seems that tophat2 doesn't read correctly the two lists of fastq files passed as lists of comma separated files. In particular it ignores the last file of the second list (the second mate)

More in detail: I have a library that has been sequenced on two lanes in paired-end mode. So I have two pairs of fastq files. For testing purposes I reduced the number of reads in each file as follows:

## Lane 3:
s_3_1.fq.gz: 25000 reads (mate 1)
s_3_4.fq.gz: 25000 reads (mate 2)

## Lane 4:
s_4_1.fq.gz: 50000
s_4_4.fq.gz: 50000

Now, if I run tophat like this:

tophat2 -o both1 -r 100 --mate-std-dev 80 --library-type fr-unstranded -G ${annotgtf} ${bwtidx} \
    s_3_1.fq.gz,s_4_1.fq.gz \
    s_3_4.fq.gz,s_4_4.fq.gz

It appears that the "left" reads are in total 74991 (25K + 50K), while the "right" reads are only 24992 (that is, only from s_3_4.fq.gz)

Here's the first line of the output

[2012-07-09 15:21:43] Beginning TopHat run (v2.0.4)
-----------------------------------------------
[2012-07-09 15:21:43] Checking for Bowtie
		  Bowtie version:	 2.0.0.5
[2012-07-09 15:21:43] Checking for Samtools
		Samtools version:	 0.1.18.0
[2012-07-09 15:21:43] Checking for Bowtie index files
[2012-07-09 15:21:43] Checking for reference FASTA file
[2012-07-09 15:21:43] Generating SAM header for /lustre/sblab/berald01/reference_data/genomes/iGenomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome
	format:		 fastq
	quality scale:	 phred33 (default)
[2012-07-09 15:22:10] Reading known junctions from GTF file
[2012-07-09 15:22:16] Preparing reads
	 left reads: min. length=20, max. length=100, 74991 kept reads (9 discarded)
	right reads: min. length=20, max. length=100, 24992 kept reads (8 discarded)

So, I tried to invert the order with which the fastq files are passed as last and second last arguments:

tophat2 -o both2 -r 100 --mate-std-dev 80 --library-type fr-unstranded -G ${annotgtf} ${bwtidx} \
    s_4_1.fq.gz,s_3_1.fq.gz \
    s_4_4.fq.gz,s_3_4.fq.gz

Again, "left reads" are ~75000 but now the right reads are ~50000 (only from s_4_4.fq.gz, now ignoring s_3_4.fq.gz):

2012-07-09 15:21:42] Beginning TopHat run (v2.0.4)
...
[2012-07-09 15:21:50] Preparing reads
	 left reads: min. length=20, max. length=100, 74991 kept reads (9 discarded)
	right reads: min. length=20, max. length=100, 49949 kept reads (51 discarded)

Running the two pairs of files separately produces the expected number of left and right reads, as well as concatenating the fastq files belonging to the same mate. So I guess the problem is not with the files themselves (which look fine to me anyway).

Any ideas what's happening?

Many thanks
Dario

[colaneri]

concatenating files before tophat?

I notice that you did not get any answer to your question, did you figure it out your self?
I have similar question

Hi
I have a RNA-seq library that has been sequenced multiple times, then I have four fastq files. My question is:
Do I need to concatenate them before alignment in tophat?
Can i just list the four files at the end of the tophat command like that?

If my files are fastq1, fastq2, fastq3 and fastq4,

and I do:

tophat -p 4 --segment-length 20 --no-novel-juncs -G /proj/seq/data/TAIR10_Ensembl/Annotation/Archives/archive-2013-03-06-09-54-25/Genes/genes.gtf -o C_ctrl_rep1_THout_6 /proj/seq/data/TAIR10_Ensembl/Sequence/Bowtie2Index/genome fastq1 fastq2 fastq3 fastq4

[dariober]

I notice that you did not get any answer to your question, did you figure it out your self?
I have similar question

Hi- No. No answer. If I correctly remember I ended up concatenating the fastq files.

If you get any insights please post on SEQanswers!

Dario

[colaneri]

tophat with multiple fastaq files

Hi guys, I was reading this thread because i felt was close enough to my question, but I could not find and answer, so here is my question.

I have 4 fastq files, all of them has been generated from the same RNA-seq library. So my question is, can I run all them together in only one tophat job?
which will be the command? do I need to list them separately with commas?

I'm wondering if I can skyp the step of merging the files in only one big fastq