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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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DNA concentrations for half of the samples are 0.1ng/ul before PCR. Can I do decontam? #142

Open sihanbu opened 7 months ago

sihanbu commented 7 months ago

Hello everyone, I have 1000 samples, but the DNA concentrations for more than 600 samples are 0.1ng/ul from Picogreen (pre-PCR). Is it necessary to identify contaminants by frequency where the DNA concentration information will be used? Will this situation skew my results from decontam?

I have the control samples. Can I identify contaminants by prevalence given this situation?

Thank you for your help!!

benjjneb commented 6 months ago

I have the control samples. Can I identify contaminants by prevalence given this situation?

Probably. I would recommend using the prevalence method instead of frequency here at least (since over half your real samples are below limit of detection).

How overlapping are the DNA concentrations measured in your real samples and the negative controls? How about read counts? How many negative controls do you have, and are they "full process" controls (i.e. blank sampling instruments taken through the whole process)? Or partial controls (like extraction/PCR controls)?

sihanbu commented 6 months ago

Thank you for the reply. I don't think I would be able to do any of the methods given this situation sadly as I also don't have enough negative controls. I only prepared one/two negative controls for DNA extraction ("full process") and one PCR control for each 96-well plate.

How overlapping are the DNA concentrations measured in your real samples and the negative controls? How about read counts?

The DNA concentrations of my negative controls (during DNA extraction) are 0.1ng/ul, which are similar to half of my samples. I have 11 controls in one sequencing run, and 5 of them have <1000 reads.

How many negative controls do you have, and are they "full process" controls (i.e. blank sampling instruments taken through the whole process)? Or partial controls (like extraction/PCR controls)?

I have 11 negative controls started from DNA extraction ("full process") and 9 PCR controls in one sequencing run. I have one/two negative controls from DNA extraction ("full process") and one PCR control in one 96-well plate.

Thanks.