Background:
First, I want to sequence fungal DNA collected from samples of over 100 individual trees. Each PCR sample was amplified with an unique barcoded combination of forward and reverse primers. This was done with the intent of pooling all samples together into one library, and later separating them via their respective barcode combinations.
Second, I am having trouble eliminating primer dimers from my PCRs. One solution is to E-gel or BluePippin to isolate the desired bands without the dimers. However, such approaches is too expensive and lengthy for this many samples.
Third, I'd like to be able to pool them together for one run, but running several samples under a bioanalyzer implied concentrations of dimers as well as the wanted sequences differed among all samples. The Bioanalyzer, and regular electrophoresis-gels also indicated species richness may also vary among all samples.
I've been told that equal molar dilutions are necessary to prevent erroneous estimations of fungal species-diversity among samples.
Questions:
1) I am separating each tree sample by an unique barcode combination to group their respective fungal community composition using said unique barcode. Instead of dilutions, is it reasonable to expect that I can analyze each individual sample's approximate fungal diversity for comparisons with other samples to develop general trends of species diversity among all samples?
2) According to the Qubit, my samples dsDNA concentrations range from 2 ng/ul to 14 ng/ul (includes primer dimers). What biases or confounding factors may be introduced with dilutions to equal molar dsDNA concentrations? Or in respect to inconsistent species diversity among all samples?
Thank you.
As per my understanding from your complete details given above.
You are planning to do Metagenomics of Fungal species isolated from different/plant.
You have amplified with the given 96 adapters by the manufacturer.
As per the manual, they says, you have to dilute all the samples in an equimolar concentration, otherwise, if u dont follow the same.
You may endup in a wrong image of fungal species among your samples.
Its better to do with Equimolar concentration, its like Reverse transcriptase PCR, you need a control. without that how could you say this fungal species is predominant or less prediminant among the test samples.
Hence, You try to take a qubit for the amplified products, and do dilution with calculations in excel sheet given by the manufacturer.
Primer dimer, need not to worry, it will be removed during size selection and other washing steps, even if it goes upto upstream process, it can be removed during bioinformatic analyses.
All the best.
Murugan N
Hello,
you need to approximately pool equimolar samples because your estimates of diversity otherwise would be biased by the rather different number of reads in your different samples. You could although subsample the samples with the higher number of reads to fit the number of reads from the lowest one (subsample multiple times at random).
Do not trust Qubit as it takes your primer dimers and potential other non specific bands into account.
Two options :
1- you can purify easily with magnetic beads (SPRI) and decrease the volume ratio beads/PCR to remove shorter products (see seqanswers forum of email me if interrested), then IF you get specific single band products then use Qubit concentration to pool at equimolarity, if needed load the pool on an agarose gel and cut the band of interrest (in the case unspecific bands appear on all of your PCRs), then sequence the pool
2- do not worry too much of the differences between PCR concentrations if you think that they are close from eachother, pool then load on agarose gel, cut the band and purify then sequence the pool.
Those are geenral guidelines, it depends on your particular PCR patterns and also on your particular biological question.
Hope this helps,
JF
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