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SE reads in fr-firststrand, fr-secondstrand

Somehow I am totally confused by the definition of #1 read, #2 read and the library types.

On the picture below, how /2 or /1 is defined (though it is PE)? What does it mean "the first read"? I though that the leftmost read is always the first and the right most in always the second read. Or does it mean the first read is sequenced?

The definition of fr- firststrand is "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)".

fr- firststrand corresponds to dUTP method, where the first read sequenced matches the dUTP strand (to original RNA) but why is it considered to be the right-most end? Why not the left-most end?

rna-seq sequencing
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updated by JourneyToAbyss JourneyToAbyss • written by tonja.r tonja.r

2 answers

fr-firststrand and fr-secondstrand refer to stranded libraries done through two different protocols

One is the dUTP method, which preserves the second strain

The other one uses the Illumina method and maybe one of the SOLiD method as well (revise) that preserves the original RNA sequence, that is the first strand

More information can be obtained after googleling, such as this

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updated by Ram Ram • written by Antonio R. Franco Antonio R. Franco

IIlumina preserves the original RNA sequence, that means, that the first sequenced read is complementary to the original RNA sequence, therefore the left-most read on the figure is labeled with /1. Ist it correct? Or why is the left-most read labeled with /1?
I do not understand the labeling of the reads in the figure and why "right-most end of the fragment (in transcript coordinates) is the first sequenced" defines the fr-firststrand.

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written by tonja.r tonja.r

I think you need to find and revise the protocols for making the stranded libraries to notice what of the two strands is preserved af the time of making the libraries, and after analyzing for a while, you will figure out yourself the answer

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updated by Ram Ram • written by Antonio R. Franco Antonio R. Franco

I did study it and I still did not figure it out That's why I posted my question here describing exactly what I do not understand.

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written by tonja.r tonja.r

I am confused, the dUTP method should preserve the first strand and destroy the second strand.

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written by xiaoleiusc xiaoleiusc

Not trying to dig up a old thread. However, I believe Antonio is incorrect. this publicaiton says dUTP degrades the second strand (thus preserves the first strand). And the image antonio posted above can be found here in a blog-post, where it lists dUTP as preserving only the first strand.

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written by JourneyToAbyss JourneyToAbyss
Notice the color of adapters being linked to the RNA You always know what adapters are being linked to the left and the right side of the RNA. And you also know whether the original strand of RNA is preserved (Illumina) or the complementary (dUTP). Since you sequence using these adaptors, you can know what side of the RNA is sequenced, therefore you can define /1 or /2 to your reads depending upon the kind of library you made
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written by Antonio R. Franco Antonio R. Franco

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