Does single end sequencing sequence both strands of the original fragment?

Question

[Steven]

Our lab has again questioned if single end sequencing does indeed sequence both strands of the original fragment, the Illumina videos do make this somewhat confusing. Can we confirm that the below diagram is indeed what happens?

In the Illumina video it only shows one strand binding the flow cell and beginning the process of clustering, but I assume both strands bind and then begin the process, but showing both strands would have made the video confusing.

My lab needs to sequence up to 250bp in one direction with an iseq (300 max cycles) and capture the sequence of both the forward and reverse strands (looking for base errors in each strand).

Answer 1

[GenoMax]

If you do single-end sequencing then only one end of the library fragment is going to be be sampled. If you have the exact DNA fragment (but now flipped 180 in a separate library fragment) then the other strand in that fragment will be sequenced.

While both p7/p5 bind at the end of the initial clustering p5 are washed away leaving only the p7 end bound strands. http://nextgen.mgh.harvard.edu/IlluminaChemistry.html should help clarify this. Also https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced/

My lab needs to sequence up to 250bp in one direction with an iseq (300 max cycles) and capture the sequence of both the forward and reverse strands (looking for base errors in each strand).

If you need sequence from both strands then you should do paired-end sequencing. Use a MiSeq if iseq only does 300 cycles max. Please remember that library fragments have a distribution of insert sizes. Not every fragment is going to be of the same size.

[Asaf]

I will just add that it depends on how you build the library - if it's shotgun then the 5' adapter will be ligated (or added in another way) to either the + strand or the - strand in a 50/50 chance (there are biases but let's forget about them). If you're doing amplicon sequencing like 16S then only one side will be sequenced since the 5' adapter will be attached to the side with the adapter sequence.

[Steven]

Hi GenoMax, thanks for the quick reply, I think the confusion comes when the double stranded DNA is first denatured giving the two strands, so each of the two single strands denatured from the original double strand (forward and reverse) are bound to the flow cell?

Then through bridge amplification both strands are amplified and become tethered in the opposite direction, so when we wash off all the p5, we still have DNA from both the forward and reverse because bridge amplification has 'crossed' them over many times.

We are sequencing synthetic DNA so are not carrying out any fragmentation of the DNA, we either ligate or PCR the adapters straight onto the DNA.

[GenoMax]

double stranded DNA is first denatured giving the two strands, so each of the two single strands denatured from the double strand (forward and reverse) are sequenced once?

Each of the strands will have either a p7 or p5 adapter. While both strands will initially bind, p5 adapter bound fragments are cleaved at a point leaving only strands bound by p7 adapter. This ensures that all copies are sequenced in the same direction. Unless you do paired-end sequencing the other end of these fragments will not be sampled/sequenced. Since you need to verify sequence on both strands you should do paired-end sequencing ensuring that both ends of same exact fragment are sequenced.

[Steven]

My original thought was what you said above, but I work in a very interdisciplinary environment and everything gets questioned lots, a picture paints a 1000 words, this is what I mean.......

Following initial bridge amplification, the remaining i7 tethered sequences are then sequenced again in the other direction for paired end.