Warning: Could not find FASTA file /home/praveenkumarr/rice_default/bwt/genome/genome.fa

Question

[PK]

Hi, I am early user of RNA Seq data analysis. When i run tophat

Warning: Could not find FASTA file /home/praveenkumarr/rice_default/bwt/genome/genome.fa
[2017-02-01 16:46:40] Reconstituting reference FASTA file from Bowtie index
Executing: /opt/bowtie2_2_2_6/bowtie2-inspect /home/praveenkumarr/rice_default/bwt/genome/genome > sample9_Rep2_WTPB1/tmp/genome.fa

It says like that. In my directory i have genome.con file instead of genome.fa (/home/praveenkumarr/rice_default/bwt/genome/genome.fa) but somehow i got output (accepted_hits.bam). My question is output file valid or not if it is not valid what is proper way...please help me

[d-cameron]

What happened to the genome.fa that the index was built from?

[PK]

i guess bowtie index. comments seems to be.

[d-cameron]

Your reference genome has been moved or deleted. If such a critical file has gone missing, I would be concerned about all the other files you're intending to use in your analysis. For example, if you use annotations for a different version of the reference genome, then your gene positions will be incorrect. How will you know if your annotation coordinates match your reference genome coordinates if you don't know what version of your reference genome you are using?

[PK]

Hi, Thanks for your comment. I am using MSU7 oryza sativa as a reference file. I have file all.con instead of all.fa . The file is still there.

[javanokendo]

copy genome.fa to the directory containing the indexed genome with the same indexbase name

Answer 1

[GouthamAtla]

The output is valid.

It could not find genome.fa so it reconstructed the fasta from bowtie2 index using bowtie2-inspect

If you have genome.fa in /home/praveenkumarr/rice_default/bwt/genome/ , it would not show that warning.

[PK]

Thanks for your reply. I have another doubt. How to calculate inner distance between read of pair from .bam file which were generated by Tophat or other possible (-r 60' tells TopHat the expected inner distance between the reads of a pair. [fragment size - (2 x read length)]. 260 - (2 x 100) = 60). I went through some biostar answers but i am not able to understand.

[d-cameron]

Different versions of the SAM file specifications gave different definitions of the fragment length/insert size. Check the documentation of your tool (TopHat) to see which definition it uses and in what context.