Can the sam files from Hisat2 used as input for HTseq?

[jingyawang]

Hello, I am using Hisat2 for paired-end RNAseq, gene differential expression analysis. I am wondering can the sam files from Hisat2 directly used as input for HTseq, which I believe requires name sorted?

If not, should I convert as below?

hisat2 -x index -1 sample_R1.fastq -2 sample_R2.fastq -S sample.sam
samtools view -bS sample.sam > sample.bam
samtools sort -n sample.bam sample.sorted
samtools view -h -o sample _sort.sam sample.sorted.bam
htseq-count -m union -s no sample _sort.sam genes.gtf > sample _sort.readcount.txt

This takes quite a long time though… Is there a faster way? Thanks so much!

[GenoMax]

If you see "unsorted" in the sam header then you would need to sort the file. That said why don't you try featureCounts? It is faster and will sort the file (it does sort a BAM file and probably will sort SAM as well) as needed.