Next generation sequencing methodology

Question

[ansarruby22]

Hi there,I am new to biotechnology. It is a very basic question .for ngs my understanding is dna is fragmented randomly into pieces each fragment is ligate with adapters and then each fragment (withadapter) is amplified many times to get exact copies of each fragment .that means all (particular fragment) will have same length and same sequence of BP. If it so how we will get overlapping reads when we compare the reads with reference genome.? All reads from same fragment will have same sequence ?(if we consider single end). Please help !!!!

[Michael]

What was your question again?

[Medhat]

please take a look at this

[ansarruby22]

"Precursor labelled " do you mean fluroscent taged nucleotide will stop the polymerisation . Still not clear.

[Antonio R. Franco]

Once the tagged fluorescent part of the molecule that is stopping rhe polymeriation is detected and the base recognized, is detached from the sequencing nascent chain, and another precursor can join the game

[ansarruby22]

1. One Cluster =one read? Or many reads of same length and same sequence.? Am I right

[Antonio R. Franco]

One cluster is composed by many identical fragments that have been amplified by the special bridge method that Illumina uses.

They are a bunch of clonal (so identical) fragments.

The idea is that if you add a labelled precursor to only a single fragment of DNA attached to the flow cell, the signal you obtain will come from a single precursor being incorporated to the sequencing chain, and the device does not have a resolution enough high to measure that single label

If you amplify that fragment through the bridge method, you build up a bunch of clonal and identical fragments LOCATED at the same place.

This allow the incorporation of many labelled precursors at the same time, and theoretically in phase (the same base every time in every cycle). This amplify the signal, and the device can measure it with confidence

[jotan]

• Dot point out what you currently know about sequencing in short clear sentences.
• Dot point out the parts which are confusing you in short clear questions.

It will be easier for people to help if they can understand what you are asking.

Answer 1

[Antonio R. Franco]

You break your DNA or cDNA into million/billions of different and random pieces.

• Then, you add adapters
• Then you add to your flow cell all of these random pieces. Each piece will land in a different part of the flow cell
• So at this time, you only have in each place where the DNA has landed a single chain of DNA.
• If you would add your reagents (read labeled precursors of the DNA) at this point to ascertain the DNA sequence, only ONE chain of the DNA will be used to sequence that particular DNA. That means that only ONE labeled precursor will be added for each of the DNA fragments
• And this means that you will get a very poor signal, as only one labeled precursor will be measured in each cycle
• To overcome this, every single and individual piece of DNA, already stuck to the flow cell, are PCR amplified. In Illumina, this PCR amplification is done ina a way that you generate a cluster of DNA fragments coming from a single DNA piece to form hundreds of clonal fragments of DNA, each located at the same place
• Thus, you will be able to measure the simultaneous incorporation of many labeled precursors at the same time, then making feasible for the device to measure it

[ansarruby22]

Thank you very much Mr. Antonio. My next question is One cluster will give one read ? When we collect all reads from all the clusters and line them horizontally with help of reference genome we will get our complete dna sequence. Am I right?

[Antonio R. Franco]

One cluster gives you a read. The genome is attained after using an assemble program