Minmum amount DNA for metagenomics

[SDPA_Pet]

Hi, our lab is going to do metagenomics using Illumina MiSeq Nextera Kits. It is the first time we do this. We used to use 454 sequencing.

Has anyone know the minimum amount DNA that I need for each sample? Our samples are environmental samples. It's very hard to extract DNA.

Thanks,

[nangel]

Hi. We have also made the move over from 454 to Illumina and have environmental samples that often have limited DNA available. We have made successful libraries using the following DNA: Nextera XT 0.25ng/ul needs a total of 1ng plus 2 ul for qPCR quant, Nextera 2.5ng/ul needs 50ng + 2ul, Truseq nano- 2.5ng/ul needs 100ng + 2 ul. I hope that helps!

[SDPA_Pet]

Hi thanks.

BTW, what the difference between Nextera XT and Nextera. It seems that XT kit use much less DNA?

Are these two kits that can generate the same length of reads? generate same number of reads?

What you did is PCR amplicon sequencing (tag sequencing)? or metagenomics?

Thanks.

[nangel]

XT kit uses much less DNA but this means that there are slightly more PCR steps in the protocol (but only 12 so it's not terribly drastic)- that might be a consideration, if you had the available DNA you might argue that limiting the PCR rounds will reduce the chance of biased amplification of the library. There is also a slight difference in the clean up between the steps of the protocol but I don't think this would have an impact on the resulting libraries.

The kits generate same length and same number of reads- loaded at equimolar levels.

And yes we are using for shotgun metagenomic libraries. We also run PCR amplicon sequencing with Nextera Indexes but I thought you were looking at metagenomic libraries. If you require for PCR amplicon there are a number of considerations for starting material but we try and standardize around 5ng/ul and ask for 20ul of DNA for successful amplicon production. I could advise further if needed.

[SDPA_Pet]

Thank you.

Yes, we do shotgun metagenomics. We only extract DNA and send it to sequencing center. They will do the library part. They just tell us they use Nextra kits. I will ask them what exact kits they use.

BTW, when you said PCR, I suppose you mean bridge amplification? Right? Unlike 454, Illumina doesn't use emPCR, right?

[SDPA_Pet]

Hi, what DNA purification kits do you use for the illumina sequencing? We just purified the a plate. I measured the DNA concentration before and after purification. I found there is only 2% re-coverage. Total DNA concentration less than 1ng/ul. This is horrible.

[SNPsaurus]

The PCR steps in the Nextera protocol are PCR of fragmented DNA with adapters, while the mixed population of fragments is in solution. The amplified library is then clonally amplified on the flow cell with bridge amplification.

There are "PCR free" library methods (but those still include bridge amplification) but Nextera needs some polymerase action to add full length adapters to the nubs left by the Nextera reagent.