Question: RNA-SeQC does not calculate information about "Strand Specificity"
0
gravatar for Lina F
4 months ago by
Lina F80
Boston, MA
Lina F80 wrote:

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:

rnaseqc_strand_specificity_output

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!

rna-seq qc • 237 views
ADD COMMENTlink modified 4 months ago by Charles Warden4.8k • written 4 months ago by Lina F80
0
gravatar for Charles Warden
4 months ago by
Charles Warden4.8k
Duarte, CA
Charles Warden4.8k wrote:

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)

ADD COMMENTlink modified 4 months ago • written 4 months ago by Charles Warden4.8k

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.

ADD REPLYlink written 4 months ago by Lina F80
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 938 users visited in the last hour