Hi, may I know is there any "minimum" number of reads that we need in the sampling step in order to produce a good and reliable result in the core-metrics-phylogenetic analysis? I have a set of 16s data and my max sequence reads is 308443 and minimum is 976 before running dada2. After quality filter…

Hi @Clara , That's an important question but unfortunately there isn't a very clear and easy answer as it depends on many factors. I made an attempt at highlighting some of the factors on another thread here which might help you decide. That being said, I think for most common samples 6,000 would be…

Dear Bod, THanks a lot for the reply and the link which I never notice before asking. It is really useful. The samples that I am working with are human microbiome samples. However, I have a query here, if let's say I choose 12123 as the sampling depth, if I am right, this means that all my samples…

Hi @Clara , You're absolutely right, with the rarefying technique you are tossing away the majority of your hard-earned reads, in fact that is one of the major cited points against rarefying . However, as discussed in the papers that was linked in the other thread, the choice of rarefying or normali…

Hi @Mehrbod_Estaki Thanks again. Another thing to confirm, when you said "rarefying" here, if I am correct, you meant the --p--sampling depth in the core metric analysis and not alpha Alpha rarefaction plotting?

Correct! Rarefying refers to the act of randomly throwing away reads to a defined level which is exactly what the sampling depth option does. The alpha rarefaction plots are generally used to determine the upper limits of richness/evenness in a sample and is one method one can use to determine a 'sa…

Your target sequence depth depends on what your aims are. Last time I did 16s on gut microbiome samples, we checked a number of cutoffs after clean up. We had 3 different typed of samples, including biopsies and faecal. Our aim was targeted towards comparing the different sample types so we set o…

@Christopher_Poulton , thanks for the sharing, very useful :grinning:

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