Trinity strand specific: RF or FR
1
2
Entering edit mode
8.9 years ago
user230613 ▴ 380

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.

trinity • 15k views
ADD COMMENT
0
Entering edit mode

Was this paired end?

ADD REPLY
13
Entering edit mode
8.9 years ago
Juke34 8.8k

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.

ADD COMMENT

Login before adding your answer.

Traffic: 857 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6