Illumina PE101

[phillie]

Dear all,

I wonder what PE101 sequencing strategy means.
I think PE101 is the name of a primer/adapter.
But that pretty much all I was able to figure out what it really means...

[kwaraska]

It probably is a read type and length

Paired End 101 bases

[phillie]

It probably is a read type and length

Paired End 101 bases

Ok, but how does this work than? How do your primers determine the lenght you read?

[mastal]

Illumina PE101

the primers don't determine the read length, the amount of reagents available to the sequencer from your kits, and the number of cycles you run determine the read length.

[phillie]

the primers don't determine the read length, the amount of reagents available to the sequencer from your kits, and the number of cycles you run determine the read length.

Ok, seems logical to me, was what I was thinking.
But why do they call it PE101 than?
Is it just a name they have to the kit?
Meaning: the primers themelf could be the same as the PE91 ?

Or?

[HESmith]

Your questions indicate a fundamental misunderstanding of how the technology works. Why don't you check out the Illumina website for an explanation?

[SNPsaurus]

phillie, sequencing runs are typically SE50,100,150 or PE50,100,150 on the current HiSeq, meaning either Single End or Paired End, and then the length of the read. This can be +/- a few bases (like PE101), depending on how the operator feels about using as much reagent as possible.

So a PE101 strategy is short shorthand for saying a particular project might benefit from paired-end reads (so both sides of a DNA fragment are sequenced), that are 101 bp reads in length. That is a common approach for sequencing a genome for alignment to a reference to look for SNPs or some structural variation.

[phillie]

Your questions indicate a fundamental misunderstanding of how the technology works. Why don't you check out the Illumina website for an explanation?

I do know how it works or perhaps I am thinking I know how it works, but am wrong.

You have the single stranded dna that binds the adapters on the flow cell, then you have a primer that binds on the top of the strand and then you have the incorporation of DNA for the multiplication... after a lot of multiplications you remove all the opposite strands (the ones you are not sequencing) and then you start the sequencing by adding another primer .. so I am guessing this is primer PE101 , but still, I am confused about it.

[phillie]

phillie, sequencing runs are typically SE50,100,150 or PE50,100,150 on the current HiSeq, meaning either Single End or Paired End, and then the length of the read. This can be +/- a few bases (like PE101), depending on how the operator feels about using as much reagent as possible.

So a PE101 strategy is short shorthand for saying a particular project might benefit from paired-end reads (so both sides of a DNA fragment are sequenced), that are 101 bp reads in length. That is a common approach for sequencing a genome for alignment to a reference to look for SNPs or some structural variation.

Ok, I see what you mean.

One question: if you do the pair end strategy, I am assuming you have 1 primer binding on the "top" of the strand you are sequencing and the second on at the base of the strand (perhaps using the adapter that is attached to the flowcells as a binding place for the second primer?)

To visualise what I mean: http://seq.molbiol.ru/sch_clon_ampl.html (I use this website to understand the technique) is that in the last step: "the last operations, which are done on Cluster station are: ◦* blocking of all 3' ends (ddNTP's and terminal transferase) to prevent extension of DNA molecules on each other;◦annealing of sequencing primer" you add a second primer at the bottom too ? right?


Or do they sequence one strand, remove this one and then create a cluster with the opposite (complementary) strand and sequence this one?
So they sequence both the 5->3 and 3->5 ? Rather then sequencing just (for example) only the 5->3 direction twice (in opposite directions, 1 primer at the top and 1 ad the bottom).

[krobison]

Ok, I see what you mean.

Or do they sequence one strand, remove this one and then create a cluster with the opposite (complementary) strand and sequence this one?
So they sequence both the 5->3 and 3->5 ? Rather then sequencing just (for example) only the 5->3 direction twice (in opposite directions, 1 primer at the top and 1 ad the bottom).

Yes, that is how it works. You read in from one direction, magically flip the molecule around so you have the opposite strand and read in from that side.

(no, it's not really magic, just amazingly clever)

Typically paired end sequencing is symmetric in that you read the same length from both sides. It is not inherently this way, and some groups have effectively used asymmetric schemes.

[phillie]

Yes, that is how it works. You read in from one direction, magically flip the molecule around so you have the opposite strand and read in from that side.

(no, it's not really magic, just amazingly clever)

Typically paired end sequencing is symmetric in that you read the same length from both sides. It is not inherently this way, and some groups have effectively used asymmetric schemes.

So to summarise, its like on this picture:

First they sequence like in the picture: from the purple side to the blue side.
Then they create a new cluster, remove the ones with the blue on the bottom and sequence from the blue at the top.
Right?