I know Hisat2-StringTie-ballgown pipeline is very useful, but I'd also like to set up a Hisat2-StringTie-Cuffdiff pipeline.
Here are the my commands for each step:
$hisat2 --dta -x fruitfly_tran -1 smGSM794483_C1_R1_1.fq -2 smGSM794483_C1_R1_2.fq -S smGSM794483_C1_R1.sam
$samtools view -bS smGSM794483_C1_R1.sam > smGSM794483_C1_R1_unsorted.bam
$samtools sort smGSM794483_C1_R1_unsorted.bam smGSM794483_C1_R1
$stringtie -G fruitfly.gtf -o smGSM794483_C1_R1.gtf -l smGSM794483_C1_R1 smGSM794483_C1_R1.bam
$gffcompare -r fruitfly.gtf -G -o merged stringtie_merged.gtf
$cuffdiff -o cuffdiff -b fruitfly.fa -p 4 -L C1,C2 -u stringtie_merged.gtf smGSM794483_C1_R1.bam,smGSM794484_C1_R2.bam,smGSM794485_C1_R3.bam smGSM794486_C2_R1.bam,smGSM794487_C2_R2.bam,smGSM794488_C2_R3.bam
And in the final step of cuffdiff,
I saw some errors:
BAM record error: found spliced alignment without XS attribute
I noticed that some parameters in the pipeline may be not good for cuffdiff.
In the hisat2, I should use --dta-cufflinks other than --dta ...
Would you please give me some suggestions?
Thanks so much for your help!!