Question: I have got some RNAseq data from Mus Musculus using TruSeq Stranded Total RNA in paired-end. I want to count my reads over my genes regarding to the gene orientation. As if a gene is plus strand, reads on forward strand falling into my plus gene area should be counted and if a gene is minus strand, reads on reverse strand falling into my minus gene area should be counted.

Let's take two examples from Ensembl :

- `Hmgb2` : `ENSMUSG00000054717.7` `chr8:57,511,907-57,515,999` `forward strand` http://www.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000076617;r=12:113418558-113422730
- `Ighm` : `ENSMUSG00000076617.9` `chr12:113,418,558-113,422,730` `reverse strand`
http://www.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000054717;r=8:57511907-57515999

I created my index with a [gencode reference genome][1] and I did my alignments using STAR with the `--quantMode` and [an annotation file][2] from gencode. I've got my `_Aligned.sortedByCoord.out.bam` and `_ReadsPerGene.out.tab`.

Check the strand of my 2 genes into my annotation file :

zgrep -i --color "Hmgb2" gencode.vM18.chr_patch_hapl_scaff_and_23_custom_igh_gff3sort.annotation.gtf.gz

#chr8	HAVANA	gene	57511907	57515999	.	+	.	gene_id "ENSMUSG00000054717.7"; gene_type "protein_coding"; gene_name "Hmgb2"; level 2; havana_gene "OTTMUSG00000060717.1";

zgrep -i --color "Ighm" gencode.vM18.chr_patch_hapl_scaff_and_23_custom_igh_gff3sort.annotation.gtf.gz

#chr12	HAVANA	gene	113418558	113422730	.	-	.	gene_id "ENSMUSG00000076617.9"; gene_type "IG_C_gene"; gene_name "Ighm"; level 2; havana_gene "OTTMUSG00000051485.2";

Ok, good I have `Hmgb2` plus strand and `Ighm` minus strand.

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Check the read strand under IGV for these two genes :

`Hmgb2`

`Ighm`

`Hmgb2` gets a lot of paired-read F2R1 (blue reads) and vice-versa `Ighm` gets a lot of paired-read F1R2 (red reads)

Note : As paired-end Illumina sequencing is R1 forward and R2 reverse, I was expecting R1 to lead the forward strand, which is not my case but anyway, it is not the point, R2 is leading the forward strand.

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Reading the documentation of [STAR][3], part `7.Counting number of reads per gene.`

> STAR outputs read counts per gene into ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options:

> - column 1: gene ID
> - column 2: counts for unstranded RNA-seq
> - column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes)
> - column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse)

>Select the output according to the strandedness of your data. Note, that if you have stranded
data and choose one of the columns 3 or 4, the other column (4 or 3) will give you the
count of antisense reads.

So, what I get is that, either the 3rd column give me my sense count or antisense, and the 4th will give me the opposite strand result.

Fine. But I want to know if STAR take the gene strand into account. I've found this threa [thread][4] (with $2 = column2, $3 = column3, $4 = column4)

>$2 is for unstranded hits, but those overlapping on opposite strand of features are considered ambiguous. $3 reports hits based on the strand you have given in your gff annotation, and $4 in the reverse direction of your features in gff (for PE-data the 5'3'-direction is also considered). Refer to -s option of htseq-count

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So, I was expecting to get high count of `Hmgb2` in either column 3 or 4 and high count of `Ighm` in the other column

`Hmgb2` / `ENSMUSG00000054717.7`

grep "ENSMUSG00000054717.7" file_ReadsPerGene.out.tab

#ENSMUSG00000054717.7	3400	31	3369

`Ighm` / `ENSMUSG00000076617.9`

grep "ENSMUSG00000076617.9" file_ReadsPerGene.out.tab

#ENSMUSG00000076617.9	16063	11	16052

All my high counts are in the 4th column... Did I forgot to tweak some options ?

[1]: ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M20/GRCm38.primary_assembly.genome.fa.gz
[2]: ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M20/gencode.vM20.chr_patch_hapl_scaff.annotation.gtf.gz
[3]: http://chagall.med.cornell.edu/RNASEQcourse/STARmanual.pdf
[4]: https://www.biostars.org/p/218995/

Answer: Most stranded libraries produced in the past ~7 years should correspond to the counts in the last column, wherein read 2's orientation matches that of the transcript. The description you linked to is wrong. Column 2 is for unstranded libraries, so the strand of a feature on the genome is ignored. Column 3 assumes that the first read in a pair's orientation matches that of the originating fragment (this is rarely the case) and column four the same but for read 2. TruSeq kits use the standard method, so the last column is correct.