Custom primers and PhiX on MiSeq

[gleb]

Hi all,
I have a question about using custom primers to sequence 16S rRNA using MiSeq. I have not done a run since last April and while reviewing updated manuals from Illumina today came across a troubling statement. This text was added in July 2013, and is completely new to me.

"When custom primers are used for Read 1 and Read 2, the software directs the sippers to reservoirs 18 and 20; therefore, Illumina primers are not used for the sequencing run. If Illumina primers are not used, the optional Illumina PhiX control cannot be sequenced."

The way it was previously explained to me is that with a low diversity library such as 16S, during a given cycle, all the clusters light up with the same color. This is why it is necessary to add PhiX which will provide diversity. If this is the case then PhiX needs to be sequenced simultaneously with the library. I thought that Illumina sequencing primers that are a part of the kit, get injected into the flow cell together with the custom primers that I load, which allows for PhiX sequencing.
Has anyone done 16S sequencing on MiSeq recently? Is this new to v3 kits?
Thanks in advance!

[kmcarr]

Originally Posted by gleb

Hi all,
I have a question about using custom primers to sequence 16S rRNA using MiSeq. I have not done a run since last April and while reviewing updated manuals from Illumina today came across a troubling statement. This text was added in July 2013, and is completely new to me.

"When custom primers are used for Read 1 and Read 2, the software directs the sippers to reservoirs 18 and 20; therefore, Illumina primers are not used for the sequencing run. If Illumina primers are not used, the optional Illumina PhiX control cannot be sequenced."

The way it was previously explained to me is that with a low diversity library such as 16S, during a given cycle, all the clusters light up with the same color. This is why it is necessary to add PhiX which will provide diversity. If this is the case then PhiX needs to be sequenced simultaneously with the library. I thought that Illumina sequencing primers that are a part of the kit, get injected into the flow cell together with the custom primers that I load, which allows for PhiX sequencing.
Has anyone done 16S sequencing on MiSeq recently? Is this new to v3 kits?
Thanks in advance!

Primers are only "sipped" from either the standard primer wells or the the custom (18,20) wells, never both. The solution to have both your custom primers and the Illumina standard primers (for PhiX) used is to mix your custom primers into the wells which already contain the the Illumina standard primers. Make sure your DO NOT indicate "Custom Primers" in your sample sheet or the MiSeq will try to "sip" from empty wells for the primers.

I don't know off the top of my head what the required primer concentration & volume to add are. Contact your local Illumina Field Application Scientist or Illumina Tech. Support for information on this procedure.

[gleb]

Thanks kmcarr,
Your answer is very useful. I looked it up and found the right wells to load the primers and correct settings. Started the run today.

[mcastelino]

Hi Gleb
Just wanted to know how your run fared after spiking the custom primers into the wells that hold the illumina primers?

[gleb]

Originally Posted by mcastelino

Hi Gleb
Just wanted to know how your run fared after spiking the custom primers into the wells that hold the illumina primers?

mcastelino, I had no problem, that really turned out the way to do it.

[mcastelino]

I wanted some advice on custom primers and 16S libraries. I have had unsuccessful runs on the MiSeq while using a published protocol (attached here) for V3-V4 primers (along with the adapter, indices as suggested) with an amplicon size of 450bp insert (~550bp with everything added on). The sample library was denatured and diluted to 4pM with 10% 4pM PhiX and run on version 2 and version 3 (Initial 6pM with 15% 20pM PhiX and then subsequent 2pM with 10% 20pM PhiX – after discussion with the Illumina personnel) of the MiSeq but both resulted in an over clustered run with absolutely no data. The custom primers as you did were spiked into the standard primer wells (according to the protocol) so that PhiX could be added to the sample library. I am presuming you have had a successful version 3 run. I have done an instrument check with regards to volume test which it passed, but the only claim from the Illumina is that I have to dilute my libraries further.
The quantitation and pooling was done using qPCR (KAPA kit) and Bioanalyser (high sensitivity kit) and therefore as accurate as possible. Is there a problem with interference of the spiked sequencing primers with cluster generation? Or is there something that I am overlooking?
I have had successful version 2 runs before when I have done 2 stage library prep – Amplicon PCR and then indexing/adapter ligation for similar libraries using NEBNext DNA Ultra kit.
Speaking to the University of Michigan contact - they have had problems with version 3 reagents and have stuck with V4 region for their 16S work on the version 2 reagents.
Any thoughts will be appreciated.
I am however going start using the 16S workflow (demonstrator protocol) published by Illumina for the V3-V4 region (fortunately) - but wanted to check that there is nothing more that I should do about the library itself or check about the library with regards to concentration of NaOH to use etc

[gleb]

Originally Posted by mcastelino

I wanted some advice on custom primers and 16S libraries. I have had unsuccessful runs on the MiSeq while using a published protocol (attached here) for V3-V4 primers (along with the adapter, indices as suggested) with an amplicon size of 450bp insert (~550bp with everything added on). The sample library was denatured and diluted to 4pM with 10% 4pM PhiX and run on version 2 and version 3 (Initial 6pM with 15% 20pM PhiX and then subsequent 2pM with 10% 20pM PhiX – after discussion with the Illumina personnel) of the MiSeq but both resulted in an over clustered run with absolutely no data. The custom primers as you did were spiked into the standard primer wells (according to the protocol) so that PhiX could be added to the sample library. I am presuming you have had a successful version 3 run. I have done an instrument check with regards to volume test which it passed, but the only claim from the Illumina is that I have to dilute my libraries further.
The quantitation and pooling was done using qPCR (KAPA kit) and Bioanalyser (high sensitivity kit) and therefore as accurate as possible. Is there a problem with interference of the spiked sequencing primers with cluster generation? Or is there something that I am overlooking?
I have had successful version 2 runs before when I have done 2 stage library prep – Amplicon PCR and then indexing/adapter ligation for similar libraries using NEBNext DNA Ultra kit.
Speaking to the University of Michigan contact - they have had problems with version 3 reagents and have stuck with V4 region for their 16S work on the version 2 reagents.
Any thoughts will be appreciated.
I am however going start using the 16S workflow (demonstrator protocol) published by Illumina for the V3-V4 region (fortunately) - but wanted to check that there is nothing more that I should do about the library itself or check about the library with regards to concentration of NaOH to use etc

I have not tried V3 yet. This thread was started last January... If you get over clustering, I don't know of anything you can do other than trying lower input concentrations. I have heard of people having to go lower than recommended to get to the 'sweet spot'.

[nucacidhunter]

I have not seen any trouble with V2 or V3 chemistries for 16S applications. You are already using low pM for clustering and considering the fact that over clustering is caused by overloading I would make following suggestions:
1- Review of qPCR run to make sure it was done correctly
2- Review of library electropherogram regarding sizing (better if you could post it)
3- Review of dilution procedure

[lomoens]

Hi Gleb, I would also like to try sequencing with both the Illumina and custom primers in one run. Cool that it seems to work in your hands!
Just had a couple of questions:
- Which primer concentrations and volumes did you load?
- Were there any other settings you changed?

Thanks so much in advance!

[annaatobe]

Hi, if possible I'd also like to get some advices on 16S MiSeq sequencing.
I'm planning to do a 16S rDNA on Illumina MiSeq (targeting at 16S V3-v4 region, using V3 kit, MiSeq machine booked, library quantified with Qubit & Bioanalyzer, all followed the 16S lib prep protocol found on Illumina website). I'm just thinking of issues on library loading concentration and PhiX percentage:
Illumina protocol recommends to start the first run using a 4 pM loading concentration and adjust subsequent runs approptiately, and a ≥5% PhiX control for low diversity libraries. The sequencing center I'm going to had been running 16S sequencing for 96 samples at one time, with a loading concentration of 4 pM and a PhiX percentage of 15% (they tried 2 times, 1st time 8%, 2nd time 15% and got a better result), it worked fine.
As in my case I only have 12 samples and the samples are from a different source prepared in a different lab (compared with the former 96 samples). In this case I hope to ask: would 4 pM be OK for my library loading (for much less sample number compared to 96 samples), or should I adjust it to a lower/higher concentration? Also could I ask for some suggestions on the use of PhiX in total library?
Thanks a million! Really looking forward to the replies.

[nprash]

Has anyone spiked in custom primers into the Illumina primers well in the Nextseq cartridge? Can you provide information on where the Illumina prefilled read and index sequencing primers are located on the reagent cartridge?

How did it work out? Thanks!

[RCJK]

check this out:
https://support.illumina.com/content...5057456-01.pdf

[nprash]

Thank you! I found this but it doesn't tell me where the Illumina primers are located so I can spike custom primers into them like the original poster on the Miseq. Let me know if you find this info too!

[JoeKutch]

This Illumina support bulletin lists the positions, total volumes, and final concentrations of the sequencing and index primers for all the sequencers that support custom primers.

https://support.illumina.com/bulleti...-primers-.html

So for R1 - position 20, R2 - position 21, and index mix in 22.

[nprash]

Just what I was looking for. Thanks!