How should I align my single-end fastq file?
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4 weeks ago
Simon Ahn ▴ 10

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I have this RNA-seq fastq file which is SINGLE-end.

However, I couldn't figure out these fastq files are unstranded or strand-specific.

I cannot even know if their orientation is forward or reversed...

I ran STAR alignment with command:

STAR --runThreadN 32 --genomeDir [ENSEMBL reference folder] \
--readFilesIn [fastq file name] --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts \
--outFileNamePrefix [output file name]

but when I convert the result to cpm and compare it to paper's cpm matrix, it's quite different.

What should I do?

RNAseq raw-count fastq • 255 views
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Entering edit mode
4 weeks ago

STAR doesn't care about strandedness. But since you are going to want it later on, get STAR to output gene counts, and you can tell from inspection which orientation is right.

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4 weeks ago
KoppesEA ▴ 70

When you get to calculating normalized read counts you will need to know the strand specificity for your single-end reads.

You can directly check this with ReSQC infer-experiment.py http://rseqc.sourceforge.net/#infer-experiment-py

use some code like

infer_experiment.py -r /YourDIR/YourSpecies.bed -i /BAMDIR/YourBAMalignment.bam
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