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How should I align my single-end fastq file?

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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
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updated by KoppesEA • KoppesEA • written by Simon Ahn • Simon Ahn

2 answers

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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written by swbarnes2 swbarnes2

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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written by KoppesEA • KoppesEA

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