I am very naive with the Illumina sequencing and I got the sequenced raw data and report. I am quite new with the technique and terminology. Can anyone explain what it means that was sequenced using "2x250bp paired-end reads"? To what do the numbers refer?
Good morning Ahmad. I apologize for the the late reply. The 2x250 bp paired-end sequencing in Illumina means that your sequencing is done in two sense :forward and reverse. Each sens with 250 cycles.
Paulin Sonon Have you ever done or analyzed illumina data? Because you are wrong.
Good morning Dr. Abhijeet. Yes I work with Illumina sequencing platform. I recognize that I am still at the beginning.
I know that it will be very difficult to explain this theory about Illumina 2x250 paired-end sequencing. I thought that this definition it would be the simplest one I can give to Dr. Ali.
I'm sorry.
Firstly,you should know what is paired end sequencing.I search it in illumina website.You can find it,if not,try this.
https://www.illumina.com/science/technology/next-generation-sequencing/paired-end-vs-single-read-sequencing.html?langsel=/en/
The numbers refer to your samples' size that how long would you want to check.
Limited to this,we always fragment our samples.
Hi I agree with Bin Mr 2x250 means that you got a bunch of coupled reads (2x) each of which is 250bp long. Coupled reads mean that they are actually part (or should be) of the same fragment, that has been sequenced from both ends (so Paulin Sonon is also not wrong).
If you are so unfamiliar with the technology, I strongly advice you in looking for someone able to deal with that, especcially for downstream polishing (they need to be pre-treated before using them) and analyses.
Sorry if I add text, but I'd like also to remind that this portal is (also) for providing help to people who ask, so there's no need to be impolite or rude
Dear Marco Pietrella , that’s the spirit. Thanks for your excellent explanations
Marco Pietrella if you are referring me as impolite or rude, its may be your opinion, but in science, saying wrong to wrong is neither impolite nor rude. Scientific discussions are not intended to please or displease anyone. Illumina sequencing sure is done for forward read and reverse read but it has nothing to do with sense or non-sense/reverse sense or complimentary of the amplicon fragment. And it is also not like sanger sequencing where sequencing is done in cycles. Illumina is massive parallel sequencing which is not done in cycles. So saying sequencing cycle (250 cycles is wrong). As the term itself says (2*300 bp) the bp which means length in base pair, annotating it as cycles is ofcourse wrong. Moreover, when there is very good information provided by the company itself by the means of documents and videos, asking such question without self effort of understanding is not encouraged. Because things can be made understood to person who want himself/herself to understand.
dear Abhijeet Singh i do not want to get into discussion, I am just saying that there are many ways to say something that can also be distinguished based on how they are perceived. I agree that scientific discussions are not intended to please or displease anyone but this is not the matter here (see above). For instance, I believe that "cycles" is somehow correct, since even Illumina sequencing is performed with cycles, as every base is added in a cycle that includes adding of a blocked dNTP, washing, detection, unblocking, washing. This, at least, is my view.
Nice day!
Dear colleagues,
I think that this video can explain a lot about Illumina platforms: https://www.youtube.com/watch?v=womKfikWlxM
Best regards
Abhijeet Singh,
I concur Marco's comments 100%. There are ways and ways to say one thing.
For the sake of correctness, in pair-end sequencing, both a forward and reverse reads are sequenced, and the addition of a nucleotide at each synthesis step is called a cycle. If you are using different terms in your lab, this is okay, but it doesn't necessarily mean this is the only correct name for use of terminology.
Good day!
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