MHV_A59 featureCounts

Question

[rajdeepboral00]

Hello all,

My library preparation is reverse stranded and i want to check the presence of viral mRNA in the RNASeq data. The MHVA49 strain is single stranded positive sense RNA, so should i use the reverse stranded pipeline that i have used to align for the mouse genome previously or the strandedness will change for the virus. Can anybody please explain. I had used the reverse standed protocol and got only one gene annotated. -rw-r--r-- 1 rajdeep unix 5507911079 Dec 16 13:30 sortedMHV_IC1.bam

This was the size after I used hisat2 for the alignment using the reverse stranded protocol. And in this only one gene is annotated ,the results of featureCounts is

command

featureCounts -p -T 8 --countReadPairs -a /data/sata_data/home/rajdeep/MHV/MHV_A59.gtf -s2 -o countsMHV_CC1.txt sortedMHV_IC1.bam

Result

 Load annotation file MHV_A59.gtf ...                                       ||
||    Features : 1                                                            ||
||    Meta-features : 1                                                       ||
||    Chromosomes/contigs : 1                                                 ||
||                                                                            ||
|| Process BAM file sortedMHV_IC1.bam...                                      ||
||    Strand specific : reversely stranded                                    ||
||    Paired-end reads are included.                                          ||
||    Total alignments : 62937622                                             ||
||    Successfully assigned alignments : 4 (0.0%)                             ||
||    Running time : 0.29 minutes   

Is it ok or i have done some mistake?

[GenoMax]

i want to check the presence of viral mRNA in the RNASeq data.

Is there something else in the library i.e. host or other organism?

I had used the reverse standed protocol and got only one gene annotated.

What does this mean? You are only aligning against one gene?

[rajdeepboral00]

Yes , it has mouse genome too. I want to see the annotation of the other MHV genes too but while using the gtf file , only one gene is getting annotated (the first one of the gtf file) and in the featureCounts also its showing that the gtf file has only one feature. Is it the problem of the gtf file? How will i fix this?

[GenoMax]

Sounds like a problem with the GTF file. You may want to use a simple annotation format (SAF) file instead.

[rajdeepboral00]

From where can i get the SAF file?? Will it be suitable to use in featureCounts?

[GenoMax]

You can make one up yourself. Scroll down to the end to find an example: https://www.rdocumentation.org/packages/Rsubread/versions/1.22.2/topics/featureCounts