Question: How To Determine If Library Is Strand-Specific
gravatar for predeus
7.9 years ago by
predeus1.4k wrote:

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.

paired-end strand sequencing • 5.7k views
ADD COMMENTlink written 7.9 years ago by predeus1.4k
gravatar for JC
7.9 years ago by
JC11k wrote:

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.

ADD COMMENTlink written 7.9 years ago by JC11k

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.

ADD REPLYlink written 7.9 years ago by Pascal250

Same answer as JC, written specifically for Tophat

ADD REPLYlink modified 7.9 years ago • written 7.9 years ago by Aswarren60

Thank you everybody! This is very helpful.

ADD REPLYlink written 7.9 years ago by predeus1.4k

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?

ADD REPLYlink written 7.0 years ago by Biomonika (Noolean)3.1k
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