Trinity strand specific: RF or FR
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5.8 years ago
user230613 ▴ 310

Hi there,
I know that this is probably a common and newbie question but I can't find the solution. I have a strand specific RNA-seq data, specifically first-strand. Which is the correct way to specify this in trinity, RF or FR? I've read the manual but is not clear for me...

Thanks in advance.

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Was this paired end?

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5.8 years ago
Juke34 ★ 6.3k

The Trinity documentation says:

If you have strand-specific data, specify the library type. There are four library types:

  • Paired reads:
    • RF: first read (/1) of fragment pair is sequenced as anti-sense (reverse(R)), and second read (/2) is in the sense strand (forward(F)); typical of the dUTP/UDG sequencing method.
    • FR: first read (/1) of fragment pair is sequenced as sense (forward), and second read (/2) is in the antisense strand (reverse)
  • Unpaired (single) reads:
    • F: the single read is in the sense (forward) orientation
    • R: the single read is in the antisense (reverse) orientation

The TopHat manual says:

Library Type        Examples                    Description
fr-unstranded       Standard Illumina           Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.
fr-firststrand      dUTP, NSR, NNSR             Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.
fr-secondstrand     Ligation, Standard SOLiD    Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.

To complete that, in Trinity fr-firststrand corresponds to RF and fr-secondstrand corresponds to FR.

You can have a look here: http://rnaseq.uoregon.edu. They well explain the difference between first-strand and second-strand synthesis.

You can also have look to this publication: Sequencing technologies - the next generation. Nature reviews. Genetics. 2010. doi:10.1038/nrg2626

Briefly, if you know the technology used for the sequencing you should be able to guess.

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