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benjjneb / decontam

Simple statistical identification and removal of contaminants in marker-gene and metagenomics sequencing data
https://benjjneb.github.io/decontam/
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Questions by use concentration DNA #37

Open molboss opened 5 years ago

molboss commented 5 years ago

Dear Benjamin,

A big thanks for developing of this program! This tool is very necessary!

I would be very glad if you could answer in my questions about using decontam:

  1. Need to use value quant reading - this is the value concentration of the isolated DNA or concentration pooling library? As I understand from a series of discussions, is it possible to use the concentration of amplicons to the stage of pooling samples?
  2. Can I use Ct value qPCR (SYBR green dye)? As I understand it, you need to use the concentration of DNA? (https://github.com/benjjneb/decontam/issues/31#issuecomment-433721318)
  3. I do not have a concentration value for one sample or some samples and the program gives an error (Error in isContaminant(z3, method = "frequency", conc = "quant_reading") : conc must be positive numeric.). What is the right thing to do in my cases? Make a decision proposed by Lisa (https://github.com/benjjneb/decontam/issues/36#issuecomment-443885318)?
  4. How do I get a list of taxa names taxa identified as contaminants?

Could you help me, please. Thanking you in anticipation for your help.

benjjneb commented 5 years ago

  1. You can use either concentration data from the isolated DNA, or of the constructed library before it is pooled for sequencing. See this comment for more: https://github.com/benjjneb/decontam/issues/33#issuecomment-436658460

  2. As long as that is measuring the DNA concentration, and covers most of the range of concentrations observed in your data, it should work. qPCR methods have been shown to be effective previously (see comment above).

  3. I think you can just set those values to NA and they will be ignored. If that doesn't work, just remove those samples prior to running isContaminant.

  4. isContaminant returns a data.frame in which one column contains the TRUE/FALSE contaminant calls. So you can just give that logical vector to the vector of taxa names you are working with: taxa.names[contamdf$contaminant].

molboss commented 5 years ago

Thank you for your assistance!