Problem reading in fastq files in STAR

[mtiwaridros]

Hi

I am trying to read in my fastq files into STAR for mapping.
My input data is organized as follows:
1. 2 samples
2. 3 replicates in each
3. For each replicate, a number of paired end fastq files

I have tried several methods to input but always get the error that could not open readInFile.

For instance:

/home/STAR --genomeDir /media/ --readFilesIn /media/Sample1_R1_001.fastq /media/Sample1_R2_001.fastq,/media/Sample1_R1_002.fastq /media/Sample1_R2_002.fastq --runThreadN 4

or

/home/STAR --genomeDir /media/ --readFilesIn /media/Sample1_R1_001.fastq Sample1_R2_001.fastq,Sample1_R1_002.fastq Sample1_R2_002.fastq --runThreadN 4

Here, R1 and R2 are paired-end reads (separated by space). And Sample 1 has several such paired-end fastq files (i showed here 2 only just to be succint) (separated by ,). Similarly for other samples.

Can anyone please suggest what might be going wrong?

Thanks in advance.
Best
M

[dpryan]

Try:

/home/STAR --genomeDir /media/ --readFilesIn /media/Sample1_R1_001.fastq,/media/Sample1_R1_002.fastq /media/Sample1_R1_001.fastq,/media/Sample1_R2_002.fastq --runThreadN 4

Basically you need to separate the list of read #1 and read #2 files from each other by a space and separate different samples (or parts of a run) inside each of those by commas.

[mtiwaridros]

Hi dpryan

Thanks for your input.
What you have suggested is what I am doing but it is not working.
As I mentioned previously,
Sample 1 has paired-end reads (R1 and R2) which are present as 15 fastq files (i.e. Sample1_R1_i and Sample1_R2_i, where i=1-15).
Similarly I have multiple paired-end files for other samples.

As you suggested,

separate the list of read #1 and read #2 files from each other by a space

that would mean separating R1 and R2 by a space

separate different samples (or parts of a run) inside each of those by commas

that would mean separating R1_1 and R1_2 by ,.

This would translate to (showing it for 3 paired-fastq files only for sample1)

/home/STAR --genomeDir /media --readFilesIn /media/Sample1_R1_001.fastq /media/Sample1_R2_001.fastq,/media/Sample1_R1_002.fastq /media/Sample1_R2_002.fastq,/media/Sample1_R1_003.fastq /media/Sample1_R2_003.fastq --runThreadN 4

I have also tried listing all the R1 reads (separated by ,) followed by a space and then listing all R2 reads (separated by ,) butthat also doesnot work.

On a similar note, should I run mapping for my other samples in the same command or issue a fresh command?

Thanks.
Best
M

[dpryan]

No, for a list of 3 that would mean:

/home/STAR --genomeDir /media --readFilesIn /media/Sample1_R1_001.fastq,/media/Sample1_R1_002.fastq,/media/Sample1_R1_003.fastq /media/Sample1_R2_001.fastq,/media/Sample1_R2_002.fastq,/media/Sample1_R2_003.fastq --runThreadN 4

The general idea is "R1_1,R1_2,R1_3,R1_4,...,R1_N R2_1,R2_2,R2_3,R2_4,...,R2_N".

[mtiwaridros]

Ok. Got it.
Its working now.
And should I include other samples also (Sample2, 3 etc) also in the same command or give individual separate commands for each of them.

Thanks.
M

[dpryan]

Since it outputs to a single file (at least if memory serves me correctly), run them separately so you can get each sample in a different file.

BTW, one trick is to load the genome into memory once and then only remove it when you're done. This is easiest if you're running things in a script that iterates through samples (this is the approach I take on our cluster).