RNA seq analysis using STAR+StringTie

Question

[fatima]

Hi everybody, I am trying STAR+StringTie. I have done the alignment with STAR and included the option ''--outSAMstrandField intronMotif'' while running STAR to get spliced alignments with XS strand attribute. However, when I run Stringtie I receive this error: input file stringtie cannot be found! Has anybody had the same problem or do you have an idea what I can do???

Thanks in advance. Fatima

[Devon Ryan]

Please show the full command that's producing the error, since you're making a typo somewhere.

[Matteo Schiavinato]

The answer is STRAIGHT: you're making a typo in the command.

(btw, in english-speaking countries and contexts, "FAG" might assume other meanings that transcend the nickname purpose, FYI)

[fatima]

Thanks for the replies. The command I used to run STAR:

Path to STAR --runThreadN 20 --genomeDir --outFileNamePrefix --readFilesIn --outSAMtype BAM Unsorted SortedByCoordinate --outWigType bedGraph --outReadsUnmapped Fastx --outSAMstrandField intronMotif

The command I used for stringtie:

path to stringtie/ stringtie D20PMAAligned.sortedByCoord.out.bam -G genes_ensembl_GRCh37_20150717.gtf -o D20_run1.gtf

btw, for STAR I have done 1st pass and 2nd pass, but I only included ''--outSAMstrandField intronMotif'' in the second pass and not the first one! does this cause a problem?

Thank you so much for your help. Fatima

[Matteo Schiavinato]

for STAR I have done 1st pass and 2nd pass, but I only included ''--outSAMstrandField intronMotif'' in the second pass and not the first one! does this cause a problem?

It shouldn't!

path to stringtie/ stringtie D20PMAAligned.sortedByCoord.out.bam -G genes_ensembl_GRCh37_20150717.gtf -o D20_run1.gtf

You have to check that you are in the right directory, because you're not specifying full paths but only filenames. Therefore, you must be inside the directory containing the files. However, I guess that the problem is the space between "/path/to/stringtie" and the word "stringtie".

[fatima]

Thank you, yes it was a stupid typo!!