STAR Aligner Output BAM Processing

[komalsrathi]

Hi everyone,

I am a new user of STAR Aligner. I have been using GSNAP until now, but my new group likes STAR better. I am very excited about using it and have a couple of questions too. We will be using paired end fastq files as input. I saw there are couple of really cool things with STAR:

1. Output coordinate-sorted/unsorted BAM files using --outSAMtype
2. Output read counts using --quantMode

Question 1: Does the --quantMode perform equally well to htseq-count? Are the read-counts identical in both cases?

When I was using GSNAP, after I obtained the output BAM file, I used to perform the following steps before using htseq-count:
1. samtools fixmate to fill in mate information
2. bamtools filter to keep only "reads in proper pair" and CIGAR string should indicate atleast one match i.e. 'M'

Question 2: So do I have to do these steps after I obtain the bam output from STAR?

Question 3: My command line for htseq-count is as follows with the GSNAP output:
samtools view -f 0x0002 sample.bam | htseq-count - $GTF > sample.counts

Question 3: Do I have to use '-f 0x0002' (i.e. reads mapped in proper pair) while using htseq-count on STAR output bam file as well?

Thanks a lot!