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Old 08-20-2012, 06:54 AM   #1
kanfenfen
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Default sequencing cost for Nexera library v.s. Truseq?

Hi all,

newbie here. We are using the Nextera kit to prepare libraries but the local NGS facility doesn't support it unless we fulfill 8 lanes due to cost. I'm going through the Hiseq 2000 manual trying to figure out the difference in sequencing procedure between Nextera and Truseq, but it seems they just require different primers, which look pretty cheap...? I'm wondering if anyone can shed some light on this? Also, we are looking for sequencing facilities who support small number of submission of Nextera libraries... Thank you!
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Old 08-21-2012, 03:50 AM   #2
pmiguel
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It would not be the cost of the primers per se. But that Nextera uses (or at least allows) dual indexing. This is great, but you need to have your entire flowcell do 2 eight cycle index reads (plus the attendant "dark cycles") if you want to do dual indexing for even a single lane. Whereas the single index TruSeq libraries require only a single 6 or 7 cycle index read.

We haven't done a dual indexing run on our HiSeq, so I don't know if it is a big deal or not. I am sure I will be sweating it the first time we do, though.

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Old 08-21-2012, 09:26 AM   #3
genbio64
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@pmiguel, is that standard Illumina supported protocol to run 2 dark cycles?
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Old 08-21-2012, 09:45 AM   #4
kanfenfen
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Thanks a lot pmiguel! I see... so that means I can pool fewer samples and do single index with nextera? The dual indexing feature doesn't mean too much to me cuz I don't have that many samples to pool anyway, but the ease and low DNA input of the nextera protocol is attractive... If I understand this right, I can run nextera libraries and truseq libraries on the same flow cell to get read1 (and read2) and index i7 by simply change sequencing primers (HP6 to HP10, HP8 to HP12, HP7 to HP11) and just increase 1 cycle for index read?
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Old 08-21-2012, 10:40 AM   #5
pmiguel
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Quote:
Originally Posted by genbio64 View Post
@pmiguel, is that standard Illumina supported protocol to run 2 dark cycles?
Quite a few more that that with the nextera dual indexing. Check out ECO's thread on the topic.

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Old 08-21-2012, 10:43 AM   #6
pmiguel
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Quote:
Originally Posted by kanfenfen View Post
Thanks a lot pmiguel! I see... so that means I can pool fewer samples and do single index with nextera? The dual indexing feature doesn't mean too much to me cuz I don't have that many samples to pool anyway, but the ease and low DNA input of the nextera protocol is attractive... If I understand this right, I can run nextera libraries and truseq libraries on the same flow cell to get read1 (and read2) and index i7 by simply change sequencing primers (HP6 to HP10, HP8 to HP12, HP7 to HP11) and just increase 1 cycle for index read?
No need to change primers on a MiSeq. Unless I am missing something?

I have not looked into mixing single index TruSeq with Nextera.

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Old 08-21-2012, 10:48 AM   #7
kanfenfen
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oh i meant on Hiseq2000 though. Guess it's not a good idea to start using nextera when our facility is not ready for it
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Old 08-21-2012, 11:24 AM   #8
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I don't think there should be an incompatibility. We had previous generation Nextera libraries (with 12 indexes) and they ran fine on one lane of a HiSeq along with other people's standard libraries, they just had to mix the primers.

I did call Illumina first to confirm that it would work, then communicated what they told me to the sequencing center.
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Old 08-21-2012, 01:38 PM   #9
kanfenfen
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Thanks cliffbeall! I'm actually trying to do something like you said Could you fill me in a bit more on how to mix the primers? thought HP10(6) and HP12(8) were added when generating clusters on cBot... can you use different primers for different samples? plus illumina says HP10 and HP12 can be used for both nextera and truseq libraries, but I'm having a hard time trying to understand how that would work...
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Old 08-22-2012, 08:01 AM   #10
cliffbeall
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I was talking about the sequencing primers, at the time we got a different pair in the (old) Nextera kit.

In looking at Illumina's site, though, it looks like with present kits you now use the same sequencing primers for TruSeq and Nextera, so ignore my earlier post.
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Old 08-22-2012, 08:57 AM   #11
pmiguel
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Details available from Illumina's web site, once you register for a "myillumina" or "icom" account, at this link. I think. Works for me currently.

This is an area made very confusing by Illumina's unfortunate reluctance to release the sequence of these primers, or even specify the make up of each of their primer mixes. These mixes seem nearly always to carry the prefix "HP" followed by one or two digits. Since the earliest of these predates the HiSeq, I am going to guess this indicates "Hybridization Primer reagents".

HP3 and HP5 are documented as either "denaturation solution" or, less obliquely, "0.1 N NaOH" respectively. HP4 carries the documentation "For use when performing a multi-primer hybe". Not sure what it is.

All the rest seem to be sequencing primer(s). So, if I have this right, there are three basic classes of Illumina adapters, the original "paired end" (pre-TruSeq) adapters, TruSeq, and Nextera. The distal (outer-most) portions of these adapters all have to be similar or identical so that they can bind to the flowcell oligos. It is the proximate (inner-most) segments of these adapters that are the primer sites for insert sequencing.

Read1 is primed by HP1, HP6 and HP10 for original paired end, TruSeq and Nextera, respectively and cumulatively.


That is, I think all the oligo present in HP1 is also in HP6. And all the oligos in the HP6 mix are present in HP10.

"Read2" (reverse insert read) is primed by HP2, HP7 and HP11 respectively and cumulatively.

Index1 (also called "i7"?) is primed by HP8 and HP12 for TruSeq and Nextera respectively and cumulatively. I don't know what primed pre-TruSeq index reads -- if there is such a thing.

Index2 (also called "i5"?) is primed by HP8 for single read runs. It is read from the flowcell oligo for a paired end run.

Hope I got that right, corrections welcome.

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Old 08-27-2012, 02:08 PM   #12
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Thanks a lot pmiguel! Never thought these primers are mixtures...! This makes a lot more sense now.
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