uneven coverage of sequencing and rarefactions

Hello Michela,

Uneven sequencing is common on the Illumina platform. There are a few things you can do upstream in the sample preparation phase that should help with this. (Because this is a problem with sequencing, it's best to fix it before sequencing, instead of trying to deal with it downstream during analysis.)

The big thing my wet-lab colleagues did was normalize the amount of amplicon PCR product added to the Illumina sequencing run.

This is the basic workflow:
extract nucleotides -> PCR -> amplified libraries -> Illumina sequencing

They added a measurement and normalization step:
extract nucleotides -> PCR -> amplified libraries -> measure DNA concentration (Qubit, nanodrop, etc.) -> calculate needed volume to add a consistent mass of DNA -> Illumina
sequencing

Because the mass of PCR the product was more even, the reads per sample were more even. :balance_scale:

Let me know if this makes sense :thinking:

Are you and your team already doing something like this? If so, what concentrations of DNA are you measuring (ng/ml)?

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