How To Determine If Library Is Strand-Specific

Question

[predeus]

Hello all,

this might be a very basic question but I gave it some thought and don't see a satisfactory answer.

Let's say I have a FASTQ file from a sequencing experiment. How can one quickly determine if the given library is strand-specific?

On a related note, how important is it for the alignment (either Tophat or Bowtie) to specify if the library is strand-specific, for both single-end and paired-end reads?

Thank you in advance.

Answer 1

[JC]

The best way is to keep record of the sample preparation. But if you only have the Fastq file(s), you can map a few randomly selected reads (sometimes I run test with 1 million reads) and you must see a bias in the orientation, many of your reads will be f-f or r-r with the expected gene.

Using strand-specific information in mapping improves the resolution of multimapped reads and antisense overlapped genes.

[Pascal]

After mapping some reads you could also use infer_experiment from the rseqc package. It will give you fractions how many reads map on the same strand as in a supplied annotation.

[Aswarren]

Same answer as JC, written specifically for Tophat http://tophat.cbcb.umd.edu/faq.html#library_type

[predeus]

Thank you everybody! This is very helpful.

[Biomonika (Noolean)]

Similar question: Do I understand correctly that counting number of reads with flags 67 and 83 should give me strong bias over one or another when using strand-specific paired-end data?