multiplex PE in a single lane

[dora]

Hello,
We plan to try multiplex PE sequencing using the Illumina kit and
recipes, but we don't need to do a lot. We'd like to use a single lane
on a flow cell with all the other lanes containing standard PE
libraries. We have the following query:
the multiplex recipes use a different read 2 sequencing primer.
Have any of you tried this or got any experience/protocols ?

Thanks and Regards

[Lee Timms]

Yes we have done this.  

Add the read 2 sequencing primer for multiplexing in equal amounts to the standard read 2 sequencing primer.  

Multiplex primer - 10ul of primer + 2mL hyb buffer.

Add above to reagent tube 16 (R2 primer).

[dora]

Thank you Lee Timms! I have another query:
At the end of the first read sequencing, the extended sequencing
primer is removed and the Index Sequencing Primer is annealed to the
same strand. Then the index sequencing reaction wouldn't interfere
with the other lanes,right?
thanks a lot!

Dora

[dora]

while I'm not very clear... I haven't got a complete mulptiplex seq
guide except preparing samples for Multiplexed Paired-End Sequencing
and PE_seq_guide.
In the PE_seq_guide, " Hybridization Buffer (1492.5 μl) • Rd 2 PE Seq
Primer (7.5 μl)( The total volume should be 1500 μl)" was added to
reagent tube 16,. Total volume is not 2 ml as your protocol. Have you
got multiplexed paired-end seq guide? I wonder could I get it from
your assistance.
Thank you very much!


On 11月12日, 下午10时25分, Lee Timms <leeti...@gmail.com> wrote:
> Yes we have done this

> > Thanks and Regards- 隐藏被引用文字 -
>
> - 显示引用的文字 -

[Shirley Horn-Saban]

Hi,

Has anyone tried multiplexing with the RNA-seq protocol? illumina do not have an "official" protocol, though they do regard it as possible. Any inputs? And on the same line: has anyone tried the Agilent Exome sequence capture? Any inputs there?

Thanks,

Shirley.

[Lee Timms]

No the index primer should not interfere with the other lanes. The primer does flow through them however in my experience it has not annealed to the templates.  If you do encounter a problem you can always trim back the last 7 bases in the lanes that do not have multiplex samples.

[Lee Timms]

That protocol is somewhat outdated.  V2/V4 cluster reagents have the R2 primer pre-mixed at a volume of 2ml in port 16. If you are not using these versions just scale back on the volume of the multiplex R2 primer. 

If you are using the protocol in your email prepare the multiplex R2 primer as follows:

7.5ul Multiplex R2 primer

1492.5ul Hyb buffer

Total volume - 1500ul Multiplex R2 primer

Add the 1500ul of the multiplex R2 primer to your diluted standard R2 sequencing primer.

The total volume of Reagent 16 should now be 3ml.

Hope this helps

[Marie]

Actually, Illumina has an "unofficial" protocol to deal with mixing
the PE read2 and the multiplexed read2 primers. They suggest spiking
the multiplexing read2 primer directly into the premixed PE read2
primer on port 16 on the PEM without adding any extra hybridization
buffer. This way, each primer is entering the flowcell at the correct
concentration rather than being diluted by half, and Le Chatelier's
principle isn't violated. We've done this several times and its
worked very well.

Marie

On Nov 13, 7:05 am, Lee Timms <leeti...@gmail.com> wrote:
> That protocol is somewhat outdated.  V2/V4 cluster reagents have the R2
> primer pre-mixed at a volume of 2ml in port 16. If you are not using these
> versions just scale back on the volume of the multiplex R2 primer.
>
> If you are using the protocol in your email prepare the multiplex R2 primer
> as follows:
>
> 7.5ul Multiplex R2 primer
> 1492.5ul Hyb buffer
>
> Total volume - 1500ul Multiplex R2 primer
>
> Add the 1500ul of the multiplex R2 primer to your diluted standard R2
> sequencing primer.
>
> The total volume of Reagent 16 should now be 3ml.
>
> Hope this helps
>

[Lee Timms]

That would work too - good point.

We had no problems doing it the way we did.  

[dora]

Thank you all for your help! I will have a try.

Dora

> > > > > - 显示引用的文字 -- 隐藏被引用文字 -
>
> - 显示引用的文字 -

[dora]

Hi,
Sorry to bother everyone again.
Is there another way to validate the multiplexPE samle Library except
sanger sequencing? We just want to verify the same molar
concentration of each mixed samples with different indexs.
Thanks again!

[elaneyk]

Illumina have released a new qPCR protocol for library quantification
purposes that you can download through icom or you can try the method
developed by the guys in the wellcome trust sanger institute published
last year (PMID: 19034268). One is based on sybr green chemistry and
the other a taqman assay.

Elaine