Hello, everyone
I have a trouble in running 16s OTUs table.
The thing is, I have the same batch of soil samples, but when I take DNA, extracted them according to different aggregates size, divided into A and B parts. And they were sent to second-generation sequencing before and after
In principle, they must have the same attributes(OTUs), but the results of the OTU table are very strange. It seems that they are two sets of completely different samples, and almost do not share the same OTU as shown in my sketch figure. This is most likely due to different sequencing batches. BUT What should we do about this situation? I think downstream analysis should be affected largely and difficult…
I really don’t know how to deal with this situation. If you have any ideas, please tell me. (sorry, uploading my sketch figure is always failed. I don’t know how to handle it, so I just write the picture linkage here: https://photos.app.goo.gl/iwc3rLQ4ihfpUUJY9)
Thanks！！！
Your description is not clear. What is "aggregate size"? What means "sequencing before and after"? Before and after what? The sequencing batches are from the same libraries? Or libraries were generated twice?
Thank you for your advice on my post！
Maybe it's because my English is not very good, so the expression is not accurate, which makes you have doubts. Let me explain it.
1.“aggregate size” is means soil samples were passed through a stack of sieves to separate the aggregates by shaking at 200-250 rpm for 2 min, generating different aggregate fractions, for example, large macroaggregates (> 2 mm), and microaggregates (< 0.25 mm). I extracted DNA for different size soil aggregates separately, Which is A and B mentioned above.
2.“Before and after”: I'd like to describe that they didn't sequence at the same time, and it's been a few months apart, and built two different libraries
Maybe my description is still not very accurate, if you have any doubts, please tell me again
Thank you.
Why would you expect different aggregates sizes to have a similar microbial profile? What does a literature review says? I am no expert, but I can certainly believe aggregate sizes provides different enough micro-environments for developing different bacterial profiles between them.
However, for what I understand, aggregate size and sequencing are confounded - that is, macroaggregates were sequenced at one date, and microaggregates were sequenced at another date, months apart, correct? Is library preparation confounded as well? I would expect sequencing to have a minor batch effect, but library preparation can have a large impact.