I often see that 16s amplicon metagenomic microbiome projects use fancy PCR master mixes and the major protocols available online for Illumina, earth micrbiome project, and the Nature Protocol exchange all use hot start mixes. I am curious what the motivation is and if people have recommendations as to what mix would be best: hot start or not, home-brewed with native Taq (I once read that would reduce contamination from Taq solution), basic master mixes (those are much cheaper, how much additional risk is there I will sequence the wrong things?). I am trying to balance costs, quality, and risk of contamination (aren't we all, right). Thanks!
You are using V3-V4-V6 regions of 16s ? The short sequence length should not affect your decision. And also never do PCR before Qubit.
Purity>polymerase. In the future we might have smaller polymerases than we are using now and it might change everything. Unfortunately the answer is yes you have to pay higher than Taq :(. I do not know what is your sample but if your proteobacterial concentration is higher than %70 percent of whole bacteria than you may consider changing extraction protocol (if you want to filter only small amount of raw read in the end) chilling the sample before extraction (I would suggest you use bead extractors) As proteobacterial OMVs with strange small DNAs will be eliminated in chilling step before extraction.
Thanks for your answers Sibelle and Orhan. I forgot to mention, we're also targeting V3-V4. It seems like we have to invest in getting good polymerase then.
@ Orhan, I run every sample (after extraction, prior to PCR) on the nanodrop and plan to run a whole bunch of samples on the Qubit or an a plate reader or both. I am curious what specifically you had in mind to look out for when running samples on a Qubit.
Also, the DNA is from whole gut digestions / extractions, so there will be host and bacterial DNA in there, and we have no prior sense of what might be in there. I use spin column protocol and the samples have been in RNAlater in the -20 freezer for a few months.There is not much I can change at this stage in either the storage method (obviously since that ship has sailed..) or in the extraction protocol (already half way through my samples), but I am interested in learning about the possible caveats of our protocol. What do you think?
Extraction method is crucial. I have added three graphs and you can see the distribution differences. The experiments from an insect gut and the highest diversity achieved with fresh cut : chill sample+bacterial isolation buffer+metagenome kit. The least diversity and terrible isolation (read quality is not good as whole tissue extractor is used so most of the DNA isolated from host and its food) The moderate results was without chill+bacterial isolation solution+metagenome kit. We have also done higher order organism like cow,sheep and human. For human depending upon the target! the extraction and sample preparations should be carried out differently. For example if you are studying about IBD it is better get your sample by colonoscopy and try to get smear from intestine. Because most of the immunomodulatory microorganisms are tightly attaching the intestine. And also bowel movements or the digestion speed directly influced by these attaching microorganisms. You have said that your samples are in -20 so some of the extraction ways to get higher amount of OTU is not possible as whole microbiome is now mixed. I try to get fresh sample than prepare samples with biological filtering so that dividing the same sample in various fractions and each fractions has more OTUs to analyze. Remember that bioinformatics is not a magical tool ! It is always wetlab makes results shining and meaningful. If you do not perform sample fractionation your sample you might loose diversity. But I am not sure you have needed high diversity as I do not know your goal. For example the
Allisonella histaminiformans is population dependent co-pathogen. (It is not actuall pathogen but it decrease the bowel movement alot and this case favors inflammatory microorganism population rise. We have very small amount and in ordinary protocols we even do not see its presence. With sample fractionation we can evaluate its density and we may even understand why some people get constipated without any reason :) But if you are going to use also 18s biodiversity on the your meta-genomic approach you may use host blocking primers.
I have written the upper parts for your next round. Now we have came the current situation. Food and Gut metagenomic sample preparations always problematic and as the taxonomic amplicon sequencing reads (most of them has higher homology) share %65 identity. You said " we have no prior sense of what might be in there " did you check SRA database it is expanding every day. Or Did you check MG-RAST people also put there as it is an open annotation server. For omnivores
Zymo Quick-DNA Fecal/Soil Microbe Kits
For herbivores
Zymo Quick-DNA Plant/Seed
I would suggest you these two kits. Once I get a better results in water buffalo rumen samples with MN soil DNA isolation kit. I dont think you will face poor adapter ligation and sothat qubit will misguided you. Without performing assembly FastQc result will previously show you whether you would have good results or not (whether you filter a lot or not).