How to make the Reverse sequence from single forward sequences Illumina Hiseq?

Question

[teleos27]

Hello,

I am working with Illumina hiseq 2000, only single reads. I got my sequences from the EBI database:https://www.ebi.ac.uk/ena/data/view/PRJEB15445

I'm planning on doing some further analysis and for that I need pair-end sequences. I need to artificially generate the reverse reading. I am bit confused because I've read in different resources that stated that the Reverse sequence is only the reverse and in others that it is the reverse-complement. I was wondering if someone knows if the second read is only the reverse or the reverse-complement?

Thank you so much

Answer 1

[GenoMax]

You can't generate paired-end data after the fact (from single end data), if the original sequencing was not done as paired-end. If you were to create a reverse complement of the reads at hand there will be a perfect overlap between the two without spatial information that a normal paired-end read gives (see illustration below).

R1 ---------------------->
   -------------------------------------------------------------------------------
                                                           <---------------------- R2

[teleos27]

Thanks for your response. I was confused with the R2 output in pair end. I thought it was reverse complement but that is incorrect it's only the reverse. If I do the reverse of my fastq sequences for my single read data, I think that would work, or not?

[mastal511]

It's reverse complement, because in order to go in the 5' to 3' direction, R2 is read from the opposite strand.

[teleos27]

Could you help me with a link with this information. I'm very confused I found this information: https://stackoverflow.com/questions/32485654/what-do-illumina-hiseq-miseq-paired-end-reads-look-like http://thegenomefactory.blogspot.de/2013/08/paired-end-read-confusion-library.html