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-   -   16S V4 Indexing PCR - Thoughts on this gel? (http://seqanswers.com/forums/showthread.php?t=49391)

Egansbay 01-07-2015 07:57 AM

16S V4 Indexing PCR - Thoughts on this gel?
 
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Hi all

I'm running a microbiome analysis of fish gut microdlora using Illumina MiSeq 16s pipeline. I'd like to get your thoughts on my indexing PCR gel - some of the bands seem a little faint. Can band strength be an indicator of performance of the samples once pooled and sequenced?

Gel pic attached.

Would really appreciate any insights.

Thank you

Egansbay

microgirl123 01-07-2015 08:23 AM

After the second round of PCR (and cleanup), I quantify with Qubit, convert to nM, and pool equimolar amounts. This works well with 99% of my samples, regardless of how the band on the gel looks. Occasionally, I have a sample pretty much fall out.

Egansbay 01-07-2015 08:51 AM

Thanks Microgirl, by sample fall out, what happens exactly? Are there any reliable indicators of potential sample performance on the MiSeq?

microgirl123 01-07-2015 10:59 AM

I'm usually pooling 96 samples into a MiSeq run and sometimes a couple of them have very low amounts of reads. Possibly this is due to a quantification or pooling problem. Once in a while, it is so bad that I think it may be a case of miscalled indexes.

I've never noticed a link between faint gel bands and lower amounts of reads. I have, however, used samples that have no visible band on the gel and gotten the expected amount of reads back. Incidentally, what volume of your PCR product are you loading on the gel?

The only thing that I have seen reliably cause a problem is the presence of adapter (primer) dimers or more than one band on the gel.

Egansbay 01-07-2015 11:58 AM

Hi microgirl, thanks for your feedback, I am loading a total of 2.5ul of the first pc product? Is that similar to your volume for the index PCR? Hopefully the run will go well, only one way to find out! What samples are you running? Are they gut samples, and if so what concentration are you starting out with? Are you using the v4 set?Thanks for your help again.

microgirl123 01-07-2015 12:06 PM

We're a core facility so the first round of PCR is up to the investigator. I ask them to perform their initial PCR in a 50-ul volume and run 5 ul on a gel to be sure there's a single band (even if it's faint). The other 45 ul is submitted to us and cleaned up with Ampure XP - I usually elute in only 28 ul to concentrate and run 5 ul of this on a gel to be sure the cleanup didn't go horribly wrong. If there's still a band (even faint), I quantify this with Qubit and add up to 50 ng of DNA to the second reaction, which is also 50 ul.

Depending on how much DNA I was able to add, I run 5-8 cycles of PCR. Then another quick Ampure cleanup, eluting in 25 ul, and running 5 ul on a gel. I've also had better luck with the Phusion HF taq mastermix than with the KAPA taq Illumina calls for.

Egansbay 01-07-2015 12:12 PM

My only worry is that the DNA conc might be a bit low, but then it's hard to know how much is enough. I'm only adding an average of 10ng per sample into the index PCR. I'm hoping this will be enough?

microgirl123 01-07-2015 12:14 PM

Can you measure with Qubit/Picogreen/other fluorometric method or do you just have a spectrophotometer?

Egansbay 01-07-2015 12:16 PM

Yes measuring with qubit and also agilent bioanalyzer. The conc recorded from the agilent is slightly lower but qubit quite reliable as measures dsDNA only?

microgirl123 01-07-2015 12:19 PM

If I was adding 10 ng of DNA to the index PCR, I'd probably do 8 cycles. If your DNA is clean, it should be fine. I've successfully done it with much less. You don't actually need to end up with all that much to run the MiSeq! Try it with a couple of samples and see what you get!

Egansbay 01-07-2015 12:26 PM

Thanks microgirl, I'll try it, just have to trust the bands are pure microbial dna and hopefully everything will work out!

js0402 09-02-2018 09:19 PM

Good day Egansbay.

Im also doing the gut microflora using the illumina flatform. I want to ask, after you did your dna extraction does it normally produce a smearing on the gel agarose ?

Hope you can help me on this.
Thank you very much.

falconpunch 10-05-2018 09:00 AM

Quote:

Originally Posted by js0402 (Post 219901)
Good day Egansbay.

Im also doing the gut microflora using the illumina flatform. I want to ask, after you did your dna extraction does it normally produce a smearing on the gel agarose ?

Hope you can help me on this.
Thank you very much.

If you're still wondering after a month...

Generally no, you should see a tight band with any "normal" 16S amplification. This is because you're amplifying one specific DNA segment (typically the V4 area of the gene coding for 16S ribosomal RNA) and while this segment is variable in sequence (the info you want to know with the analysis), it shouldn't vary in length (what a band on a gel would show).

Hope that helps!

js0402 10-08-2018 08:07 PM

Quote:

Originally Posted by falconpunch (Post 220614)
If you're still wondering after a month...

Generally no, you should see a tight band with any "normal" 16S amplification. This is because you're amplifying one specific DNA segment (typically the V4 area of the gene coding for 16S ribosomal RNA) and while this segment is variable in sequence (the info you want to know with the analysis), it shouldn't vary in length (what a band on a gel would show).

Hope that helps!

Thank you for the reply! If possible, can I know the primer and pcr condition that you've been using ?


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