RNA-SeQC does not calculate information about "Strand Specificity"

Question

[Lina F]

Hi all,

I recently ran RNA-SeQC on yeast RNAseq data to assess the quality of the sequencing run.

Unfortunately, the tool returned only "NA" for the "Strand Specificity" section:

This sample contains single-end reads that I mapped to the reference using STAR using the following command:

$> ~/STAR --runThreadN 20 --genomeDir ./yeast_index --outFileNamePrefix ./star_out --readFilesIn sample_10-b-8.fastq --limitBAMsortRAM 1207105173 --outSAMtype BAM SortedByCoordinate --outSAMattrRGline ID:HWLCLBGXZ_1_17 SM:Sample_10-b-8

Are there any other options I need to set in STAR to provide strand specificity information?

Thanks for any suggestions!

Answer 1

[Charles Warden]

You can see Infer-experiment.py, is strand-specific? for more information about getting strand-specific information from RSeQC via infer_experiment.py. I'm not familiar with the report format that you provided, but I know that infer_experiment.py works. You can also double-check that chromosome format for the gene annotations matches the chromosome format of your alignment.

You don't have to specify a parameter for the strand for the STAR alignment, but you could use some or all of the following strategies to affect your splice junctions:

1) You can add --outSAMstrandField intronMotif to add a strand attribute, which might be needed for some downstream analysis

2) You can use a --twopassMode Basic setting to refine alignments around junctions

3) You can provide genomic annotations when creating your indexed reference (and there are parameters to specify a splice junction database during alignment)

[Lina F]

Thanks for the suggestion!

I am actually using the RNA-SeQC tool from the Broad Institute; that's where the report came from. I will take a look at the tool you mentioned, maybe it will be more appropriate.