Using STAR aligner output for Htseq-count and Cufflinks

Question

[candida.vaz]

Dear All,

I am using STAR aligner for mapping my paired end reads using the following parameters:

/cluster/apps/x86_64/packages/STAR_2.5/bin/STAR --runThreadN 4 --genomeDir /home/candidav/STAR-Gallus-gallus-nv5/Gallus-gallus-genome-dir-nv5 --readFilesIn /home/candidav/S1/RCI001-forward_paired.fastq /home/candidav/S1/RCI001-reverse_paired.fastq --outFilterType BySJout --outFilterIntronMotifs RemoveNoncanonical --outSAMtype BAM Unsorted SortedByCoordinate --outFileNamePrefix /home/candidav/Biomin-S1-RCI001-star-out/RCI001

Question1: Is this fine? or do I need to mention any other parameter?

Question2: I get these two output bam files, can I use them for the following downstream quantification tools?

• RCI001-Aligned.sortedByCoord.out.bam : For Cufflinks

• RCI001-Aligned.out.bam : For Htseq

Thanks in advance!

Answer 1

[Devon Ryan]

1. That's a perfectly acceptable command.
2. You don't need the file for htseq-count (featureCounts is much faster), you can use the same file as cufflinks.

As an aside, don't use cufflinks, use stringTie instead.

[candida.vaz]

Hello Devon, Thanks for your answer! I was wondering what is feature count? I am using Htseq-count. http://www-huber.embl.de/HTSeq/doc/count.html

While running STAR --outSAMtype BAM Unsorted SortedByCoordinate we get two BAM files

STAR manual recommends to use the output Unsorted bam file for HTseq. My collaborators want me to use cufflinks, so can I use the SortedByCoordinate BAM file for quantification by cufflinks?

I have to learn how to use stringTie --thanks for recommending it.

Regards, Candida