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Old 07-28-2014, 11:59 PM   #1
BioGenomics
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Default 16s MiSeq Illumina library concentration and PhiX

Hi all,

I have 2 small questions concerning the 16s sequencing protocol for MiSEq:

1) Some samples have very low concentration so we are considering loading a 2pM library instead of the more standard 4pM. Anyone having bad (low) clustering doing this or no problem when using the v3 MisEq kit (2 X 300 bp) ?

2) There are various older threads discussing the PHIX % to be added to the library. The protocol says 5 % (v3 kits again), but the rep says 10-20 %. Any comments on this ?

thanks

Greg
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Old 07-29-2014, 01:35 AM   #2
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Quote:
Anyone having bad (low) clustering doing this or no problem when using the v3 MisEq kit (2 X 300 bp) ?
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.

Quote:
The protocol says 5 % (v3 kits again), but the rep says 10-20 %. Any comments on this ?
Illumina's current official recommendation with the latest software is 5% PhiX spike-in, but 10% spike-in increases the likelihood of successful run.
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Old 07-29-2014, 04:37 AM   #3
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Thanks for this nucacidhunter. So is 4 pM the optimal (clustering) conc for amplicon Seq ?

cheers
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Old 07-29-2014, 05:42 AM   #4
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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. Final pM concentration for clustering varies among labs as the quantification systems and practices differ. For amplicons less input improves PF% and also quality scores which usually is achieved with cluster densities below 1k. Illumina also recommends incubation of HT1 diluted denatured libraries at 96°C for 2 min before loading to improve clustering GC rich fragments
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Old 07-30-2014, 12:20 AM   #5
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I had another question concerning low cleaned amplicon concentration before indexing PCR. Can the Nextera indexing PCR cycle (8 cycles) be increased (to 10-12) without any issues to increase the concentration before final bead clean-up ? Also, what's the contamination likelihood using a SpeedVac ?

cheers

Last edited by BioGenomics; 07-30-2014 at 12:50 AM.
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Old 07-30-2014, 01:59 AM   #6
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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.
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Old 08-11-2014, 04:47 AM   #7
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Quote:
Originally Posted by nucacidhunter View Post
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.

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Old 08-11-2014, 05:04 AM   #8
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Yes, 4 nM. Thanks for correction.
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Old 08-11-2014, 05:16 AM   #9
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Quote:
Originally Posted by nucacidhunter View Post
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.

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Old 08-12-2014, 02:24 AM   #10
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Quote:
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.
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Old 11-12-2014, 11:12 PM   #11
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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
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Old 12-10-2014, 08:27 AM   #12
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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/...th.1270-S1.pdf
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Old 12-10-2014, 08:04 PM   #13
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We use the NextSeq protocol with the 200mM Tris pH 7 all of the time for our MiSeq runs.
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Old 02-18-2015, 08:52 AM   #14
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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.
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Old 02-22-2015, 06:58 AM   #15
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I usually make 700 uL and load 600 uL. I have never tried less.
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Old 02-22-2015, 06:59 AM   #16
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Are you trying to go lower then 100 pM?
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Old 02-23-2015, 06:38 AM   #17
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Hi
The concentration is 150pM.
Thanks.
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Old 02-24-2015, 07:24 AM   #18
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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.

Last edited by acockburn; 02-24-2015 at 07:27 AM.
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Old 03-03-2015, 05:49 AM   #19
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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.
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Old 10-11-2017, 10:10 AM   #20
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Default 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!!

Quote:
Originally Posted by acockburn View Post
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.
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