Hi everybody! I am using RNA-Seq protocol for identifying differentially expressed lncRNAs. I have used the reference genome from gencode: gencode.vM20.lncRNA_transcripts.fa I have build the index and then run hisat2:
> hisat2 --dta -q -x mm10_lncRNA_genome -U C-P1_28454_ACAGTG_trimmed.fq.gz -S C-P1_54_L4.sam
Then I have converted sam files to bam, and then sorted them and created the bai index.
> samtools view -bS -o C-P1_54_L4_lncRNA.bam C-P1_54_L4_lncRNA.sam > samtools sort -o C-P1_54_L4_lncRNA_sorted.bam C-P1_54_L4_lncRNA.bam > samtools index -b C-P1_54_L4_lncRNA_sorted.bam C-P1_54_L4_lncRNA_sorted.bai
Finally I have tryied to use stringtie with the gtf file which is also available in gencode for lncRNA: gencode.vM20.long_noncoding_RNAs.gtf
But when running stringtie I have a WARNING mesage:
> stringtie -G gencode.vM20.long_noncoding_RNAs.gtf -l C-P1_54_lncRNA_sorted -B -C C-P1_54_lncRNA_cov.gtf -o C-P1_54_lncRNA_transcripts.gtf -A C-P1_54_lncRNA_gene-abundance.tsv C-P1_54_lncRNA_sorted.bam WARNING: no reference transcripts were found for the genomic sequences where reads were mapped! Please make sure the -G annotation file uses the same naming convention for the genome sequences.
I don't understand why i am having this problem as I am using both reference files (.fa and .gtf) from the same source.
Can someone help me?
Thanks in advance,