RNA-SEQ data, reads count zero

Question

[wschen97]

hello, everyone, I am new to RNA-seq data analysis, when I run htseq-count, in the count file I get seems all gene were count as zero, but those files do have high alignment rate(~95%), what could cause this problem? Thanks a lot for yuor kind help!

Here are some examples in those count file:

ENSG00000282815 0
ENSG00000282816 0
ENSG00000282817 0
ENSG00000282818 0
ENSG00000282819 0
ENSG00000282820 0
ENSG00000282821 0
ENSG00000282822 0
__no_feature 20452182
__ambiguous 0
__too_low_aQual 0
__not_aligned 1092920 __alignment_not_unique 1409855

here is the result of samtools flagstatus: 25007090 + 0 in total (QC-passed reads + QC-failed reads)
2052133 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
23914170 + 0 mapped (95.63% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

Answer 1

[EagleEye]

I guess the terminology for chromosome from GTF/GFF file and the BAM/alignment file is different. Example: Chromosome numbers can be "chr1, chr2, chr3..." or "1,2,3...". Changing the chromosomal terminology in GTF/GFF matching with BAM/alignment file might resolve this issue (if it looks different in both place).

[wschen97]

thanks! I will try it.

[wschen97]

hey, I have checked chromosomal terminology in GTF file and BAM file, they seems to be the same.what else problem could it be ?

[EagleEye]

Hi,

Just to know problem, try quantifying with featureCounts from Subread, http://bioinf.wehi.edu.au/featureCounts/