flipped BAM files in IGV after STAR aligner

Question

[veronicam]

Hi!

I am using the STAR aligner to align my RNA-seq data to hg38, and my outputs are all inverted when I visualize them in the IGV (they all go to the opposite direction to the gene)

RNA-seq library prep was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina

I am using the following parameters:

STAR --runThreadN 12 \ 
--genomeDir /lcrb/Alumni/Ofri/genome_directory \ 
--readFilesCommand zcat \
--readFilesIn ${name}.fastq.gz \
--quantMode GeneCounts\
--outSAMtype BAM SortedByCoordinate --outFileNamePrefix ../STAR_OUT_readStrand/${name} ; done

Any leads for why do I see al my reads inverted?

Thanks!!

[Ram]

It looks like you're using a loop. Please show us your full code, including the loop,

[swbarnes2]

Isn't that how that library prep is supposed to make them? Reverse as compared with the transcript?

[rfran010]

I agree with this. That NEB kit produces "reverse stranded" reads.