SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Comparison between SOLiD, Illumina MiSeq and Illumina HiSeq NGS_New_User SOLiD 0 12-12-2012 11:37 AM
bowtie command line for Illumina Hiseq 2000 with Illumina 1.5+ quality encoding files rworthi Illumina/Solexa 4 09-28-2011 11:25 AM

Reply
 
Thread Tools
Old 05-24-2013, 01:39 AM   #1
phillie
Member
 
Location: Earth

Join Date: Jul 2012
Posts: 16
Default Illumina PE101

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...
phillie is offline   Reply With Quote
Old 05-24-2013, 05:10 AM   #2
kwaraska
Senior Member
 
Location: Boston,MA

Join Date: Nov 2008
Posts: 122
Default

It probably is a read type and length

Paired End 101 bases
kwaraska is offline   Reply With Quote
Old 05-24-2013, 05:14 AM   #3
phillie
Member
 
Location: Earth

Join Date: Jul 2012
Posts: 16
Default

Quote:
Originally Posted by kwaraska View Post
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?
phillie is offline   Reply With Quote
Old 05-24-2013, 06:04 AM   #4
mastal
Senior Member
 
Location: uk

Join Date: Mar 2009
Posts: 667
Default 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.
mastal is offline   Reply With Quote
Old 05-24-2013, 07:21 AM   #5
phillie
Member
 
Location: Earth

Join Date: Jul 2012
Posts: 16
Default

Quote:
Originally Posted by mastal View Post
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?
phillie is offline   Reply With Quote
Old 05-24-2013, 08:38 AM   #6
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 509
Default

Your questions indicate a fundamental misunderstanding of how the technology works. Why don't you check out the Illumina website for an explanation?
HESmith is offline   Reply With Quote
Old 05-24-2013, 08:53 AM   #7
SNPsaurus
Registered Vendor
 
Location: Eugene, OR

Join Date: May 2013
Posts: 521
Default

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.
__________________
Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com
SNPsaurus is offline   Reply With Quote
Old 05-24-2013, 08:57 AM   #8
phillie
Member
 
Location: Earth

Join Date: Jul 2012
Posts: 16
Default

Quote:
Originally Posted by HESmith View Post
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 is offline   Reply With Quote
Old 05-24-2013, 08:59 AM   #9
phillie
Member
 
Location: Earth

Join Date: Jul 2012
Posts: 16
Default

Quote:
Originally Posted by SNPsaurus View Post
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).

Last edited by phillie; 05-24-2013 at 09:18 AM.
phillie is offline   Reply With Quote
Old 05-24-2013, 09:58 AM   #10
krobison
Senior Member
 
Location: Boston area

Join Date: Nov 2007
Posts: 747
Default

Quote:
Originally Posted by phillie View Post
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.
krobison is offline   Reply With Quote
Old 05-24-2013, 10:06 AM   #11
phillie
Member
 
Location: Earth

Join Date: Jul 2012
Posts: 16
Default

Quote:
Originally Posted by krobison View Post
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?
phillie is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 10:46 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO