Paired - end sequencing

Question

[adarsh_pp]

Hello,

This might be a simple doubt, but I am confused for a longer time.

In paired-end sequencing, it is said that a fragment is read in both direction. So does it mean that a single DNA strand from the double strand is read two times in both directions? or is it like reading the forward strand and then reading the reverse strand.

So in the final file what is Read1 and Read2 considering the original DNA fragment? Both these should contain same orientation of bases right?

Please give me some explaination.

Thank you

[Asaf]

Search youTube for videos on how Illumina sequencing works, once you understand the process you'll understand the data better.

[Prash]

Dear Adarsh Apart from the wonderful suggestions as above, I suggest you count the reads and the size and check the first few lines

If you are a linux user, you can use "head filename_R1.fastq" and head filename_R1.fastq

Likewise count the lines so that you ar eable to get the same reads: "cat filename_R1.fastq | wc -l"

Again, sometimes you may not get the same read lines an dcould be checked after trimming.

Hope this helps, Prash

Answer 1

[liorglic]

R1 and R2 are usually in forward and reverse orientation, respectively. That means that they come from two different strands of the double-stranded fragment. However, in most cases (genomic DNA) you don't know if the original fragment came from the plus or minus strand, so you don't really need to care about any of that. Also, software tools that process NGS data (e.g. read mapping tools) will usually try both forward and reverse orientations, so again, you don't need to worry about that.
As suggested, it can be useful to watch a video explaining the Illumina sequencing process. E.g. this one.