[sriman]

Neutralization Buffer

Hello,

I came across acockburn's suggestion on using the protocol published in the Supplementary Protocol 12: Modified hybridization buffers of the following paper: A large genome center's improvements to the Illumina sequencing system for sequencing low concentrated libraries.

My question is on preparing the neutralization buffer, it is not clear to me whether I should add the 1 M Tris-HCl pH7.3 in the neutralization buffer or is this only added to the HT1?

Please let me know if anyone has prepared this buffer before.

Thanks a lot!!

Assuming you want to load 10 pM, 1% spike in of PhiX and using the above subnanomolar protocol.

Add the following --- Volume (uL)
0.15 nM sample DNA --- 52
0.2 N NaOH --- 52

Denature 5 min.

Add 20 uL of Tris-HCl pH 7.3 to Hyb buffer.

Add the following --- Volume (uL)
Denatured DNA --- 104
Neutralization buffer --- 208

Add a volume of Tris-Hyb buffer to the desired final loading concentration which is assumed to be 10 pM, so factoring in the PhiX.

Add the following: --- Volume (uL)
Denatured/diluted library --- 311.11
Chilled Hyb buffer --- 458.89

Check pH with indicator paper. It should be between 7 - 8.5.

FINAL LOAD LIBRARY
Add the following: --- Volume (uL) Final Concentrations
10.1010101 pM Denatured Lib --- 693 -- 10 pM
10 pM PhiX Library --- 7 -- 0.1 pM


Load 600 uL into cartridge.

[rpm27]

Thank you. What I ended up doing is to combine my low concentration libraries with phix to generate a 2nm library. I do not need lots of reads and merely for verifying sequence. It still failed because of over clustering. I feel as my SybrG assay was not that accurate. I am going to run digital pcr to get more accuracy.

[acockburn]

Assuming you want to load 10 pM, 1% spike in of PhiX and using the above subnanomolar protocol.

Add the following --- Volume (uL)
0.15 nM sample DNA --- 52
0.2 N NaOH --- 52

Denature 5 min.

Add 20 uL of Tris-HCl pH 7.3 to Hyb buffer.

Add the following --- Volume (uL)
Denatured DNA --- 104
Neutralization buffer --- 208

Add a volume of Tris-Hyb buffer to the desired final loading concentration which is assumed to be 10 pM, so factoring in the PhiX.

Add the following: --- Volume (uL)
Denatured/diluted library --- 311.11
Chilled Hyb buffer --- 458.89

Check pH with indicator paper. It should be between 7 - 8.5.

FINAL LOAD LIBRARY
Add the following: --- Volume (uL) Final Concentrations
10.1010101 pM Denatured Lib --- 693 -- 10 pM
10 pM PhiX Library --- 7 -- 0.1 pM


Load 600 uL into cartridge.

[rpm27]

Hi
The concentration is 150pM.
Thanks.

[acockburn]

Are you trying to go lower then 100 pM?

[acockburn]

I usually make 700 uL and load 600 uL. I have never tried less.

[rpm27]

Hi,
I am wondering for subnanomolar concentration library, what was the final volume you (acockburn) loaded on the sequencing machine? The miseq protocol says 600ul, but curious what others have tried loading. Thank you.

[RCJK]

We use the NextSeq protocol with the 200mM Tris pH 7 all of the time for our MiSeq runs.

[acockburn]

For subnanomolar libraries we use the protocol found in the supplemental section (Protocol 12) in the paper below. Its a fantastic protocol and I have used it for libraries with 100 pM (diluted to 4 pM for the final load) and had successful runs.



Nature Methods 5, 1005 - 1010 (2008)
Published online: 25 November 2008 | doi:10.1038/nmeth.1270

A large genome center's improvements to the Illumina sequencing system

http://rdcu.be/bNuQ

Supplementary info;
Supplementary Protocol 12: Modified hybridisation buffers

http://www.nature.com/nmeth/journal/v5/n12/extref/nmeth.1270-S1.pdf

[BioGenomics]

Hi nucacidhunter et al, anybody has experience with using a 0.5 nM 16s library concentration (instead of 2nM) on the MiSeq ? The NextSeq500 has this protocol (updated October 2014, so more recent) and we thought to add the 200mM TRis buffer Ph 7.0 too to hydrolyse the higher NaOH concentration needed initially. Thanks

[nucacidhunter]

I wish Illumina would bear down and create a denaturation procedure that doesn't require a subsequent 50X dilution of the sample with buffer prior to loading.

It seems that Illumina looking into different methods for denaturing libraries for clustering. For HiSeq X system, library is mixed with three solutions (PCX1-3) and RSB (without an initial denaturation) and loaded into cBot. Libraries for this system must be denatured in cBot perhaps by heating (if someone have the related cBot recipe can check this). Also, llumina’s current protocol for clustering and sequencing on NextSeq 500 includes a naturalisation with Tris-Cl pH7.0 after NaOH denaturation step.

[pmiguel]

We saw inconsistent cluster density. Illumina recommends concentration by SpeedyVac. Other options would be concentrating them using column or AMPure beads by reducing elution volume.

Yes.
The real issue here is probably the concentration of NaOH in the sample loaded into the cassette. If it is too high, then it would interfere with binding of the amplicons to the flowcell.

I wish Illumina would bear down and create a denaturation procedure that doesn't require a subsequent 50X dilution of the sample with buffer prior to loading.

--
Phillip

[nucacidhunter]

Yes, 4 nM. Thanks for correction.

[pmiguel]

Illumina’s guide for clustering libraries on MiSeq can be find by google searching “Preparing Libraries for Sequencing on the MiSeq”. 4 pM is the concentration for denaturation step.

You mean 4nM.

--
Phillip

[nucacidhunter]

If concentration of amplicon PCR after clean-up is 200 pg/ul, using 5 ul in index PCR will result in over 200 ng amplicon which after clean-up will be at concentration of 4ng/ul. This amount will be equivalent to 12 nM (if you are using Illumina validate region primers). If you are not obtaining these figures, the problem might be in input DNA. Increasing cycle number may cause biases in amplification of various species DNA present in sample. In any case the metagenomics profile is always one view of community and there is no common agreement on it. If increasing amplicon concentration is the aim, it can be achieved easily by decreasing elution volume from beads.

I do not know the likelihood of cross contamination in SpeedyVac (I think pretty low), but it should not matter because the amplicons already have been indexed.