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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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is "quant_reading" DNA concentration in some units or total DNA #127

Open Rob-murphys opened 1 year ago

Rob-murphys commented 1 year ago

I am confused if quant_reading in the introduction is total DNA or DNA concentration in some units. If so are there any specific units?

I have the issue of having some samples which did not have enough DNA for sequencing before pooling. So I re-PCRed those samples and pooled them all or partially together and am wondering how I then give a final value to decontam for my concentrations?

benjjneb commented 1 year ago

The units don't matter as long as its the same units throughout. In other words, only the relative DNA concentrations are used by decontam, not the absolute values.

Rob-murphys commented 1 year ago

Okay thank you :)

What should I do for samples when I had to pool PCRs? (A stupid question I know but I can't find a solid answer on how to handle that)

benjjneb commented 1 year ago

What should I do for samples when I had to pool PCRs?

At what step are you measuring DNA concentrations? If you measured before you pooled separate PCRs into a common sample, then sum those measurements. If you measured after pooling separate PCRs into a common sample, then use as the measured DNA concentration as is.

Rob-murphys commented 1 year ago

I measured before pooling but when pooling I did not always take the same volume. For example it may have been 40 ul of one and 8 ul of another to make the total DNA reach the acceptable sequencing threshold. Can I still just sum them?

benjjneb commented 1 year ago

However you did your sample combination, you want the number of the combined sample to represent the same type of number as you measured in the other samples. Assuming these are all in terms of DNA concentrations, then I guess your combined samples are some sort of weighted average of the concentrations in the original samples that were combined.