I want to see the community structure of the soil amended with biochar, however, I've got difficulty in extracting high DNA yield. My samples did not pass the quality control for next generation sequencing analysis, so I'm thinking for another option with the same target yet with low conc. of DNA. Do you have any suggestions on what to do with my DNA with very low conc (>5ng/ul but less than 15ng/ul) ? Thank you very much.
I take it you are sending the samples to a commercial facility with fixed standards for acceptable material? We sequence some extremely low biomass samples that come from extreme environments--some work, some don't. If you take the risk that they won't work, would your facility make the attempt?
Someone may suggest using whole genome amplification, but I don't advise it. Your results almost certainly will be biased.
Do you need whole genome? How about just PCR the 16S up, and you'll learn who's there. That has its biases too, but far less bias than whole genome amp.
Usually if you are dealing with soil samples for any meta-genomics analysis, you need good quality of your DNA samples.
I would advise that do not waste your time trying to deal with low concentration DNA!. You may consider optimizing your DNA recovery/extraction using conventional methods or other commercial DNA extracting kits! Nowadays there are many excellent kits to help to extract total microbial DNA from soil.
-Abdelahhad
+HIllary G. Morrison, thank you for the information. I sent it for next generation sequencing analysis however it didn't pass due to low conc. of DNA. 16s rRNA gene amplification was successful yet the band is very faint, not enough for the NGS analysis. I will not test for whole genome sequencing just the V3 and V4 region of 16s rRNA gene. How would I deal with this one? Thank you.
+John Schloendorn, I have PCR product yet it is very faint and could not pass the quality control for NGS. I'm not into whole genome sequencing, just want to see the community structure of bacteria in the soil receiving various treatments (eg. biochar). Thank you sir.
+Abdelahhad Barbour, I have tried lots of protocol from published journals, I am using Fast DNA Spin kit for soil for my extraction, yet I got a very low concentration of DNA. The soil is treated with biochar which I highly considered the reason why the soil is so recalcitrant during extraction. Thank you for nswering sir.
.
Dear Angelbert Cortes,
I think Abdelahhad Barbour has rightly pointed out about the risk of working with LOW concentrations of DNA. Yes. off course, if you go for sequencing or related molecular biology works with very low yield of DNA, you may have to face problems. Better, you do some paper works and standardise the protocol for DNA isolation and estimation. There are also kits in the market which may assist you in the field of molecular biology.
Good Luck!
Hi Angelbert
you can see this paper "Assessment of Soil Microbial Community Structure by Use of Taxon-Specific Quantitative PCR Assays"
http://aem.asm.org/content/71/7/4117.full
And this "Driving forces of soil bacterial community structure, diversity, and function in temperate grasslands and forests"
http://www.nature.com/articles/srep33696
And "Plant diversity predicts beta but not alpha diversity of soil microbes across grasslands worldwide"
http://onlinelibrary.wiley.com/doi/10.1111/ele.12381/abstract;jsessionid=2C27AC0F7FED087E35CE5A332EC72F0E.f02t03
And "Soil Parameters Drive the Structure, Diversity and Metabolic Potentials of the Bacterial Communities Across Temperate Beech Forest Soil Sequences"
http://link.springer.com/article/10.1007%2Fs00248-015-0669-5
And "A determination of protective microhabitats for bacteria introduced into soil"
https://academic.oup.com/femsle/article-lookup/doi/10.1111/j.1574-6968.1991.tb04699.x
Also this "Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species"
http://aem.asm.org/cgi/ijlink?linkType=ABST&journalCode=jb&resid=172/8/4238
This "Bacterial phylogenetic diversity and a novel candidate division of two humid region, sandy surface soils"
http://aem.asm.org/external-ref?access_num=10.1016/S0038-0717(03)00124-X&link_type=DOI
This "Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities"
http://aem.asm.org/external-ref?access_num=10.1016/j.mimet.2004.02.003&link_type=DOI
This "soil microbial diversity under long-term organic and conventional farming"
http://dx.doi.org/10.1038/ismej.2014.210
this "Direct and indirect effects of tree diversity drive soil microbial diversity in temperate deciduous forest"
http://dx.doi.org/10.1016/j.soilbio.2010.05.030
This 'Estimates of soil bacterial ribosome content and diversity are significantly affected by the nucleic acid extraction method employed"
http://dx.doi.org/10.1128/AEM.00019-16
This "Short pyrosequencing reads suffice for accurate microbial community analysis"
http://dx.doi.org/10.1093/nar/gkm541
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