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Hi all, I would like to know if there is a way to calculate how many different genomes you sequence when you prepare amplicons from human gDNA. The goal is to do a power-calculation to understand how much we need to sequence a sample to identify mosaic variants with low mutant allelic frequencies.
In a pilot experiment, we have prepared amplicons from 25 ng of template gDNA. Based on an average human diploid cell DNA content of 6.5 pg, this corresponds to ~3,846 copies of diploid genomes. To the resulting amplicon was then added P5 and P7 by PCR and sequenced on a MiSeq. Will my sequencing library have covered all the genomic copies used as template? Is there a way to calculate how many I have covered? Is there a library preparation step to control this parameter?
Thank you very much!
In a pilot experiment, we have prepared amplicons from 25 ng of template gDNA. Based on an average human diploid cell DNA content of 6.5 pg, this corresponds to ~3,846 copies of diploid genomes. To the resulting amplicon was then added P5 and P7 by PCR and sequenced on a MiSeq. Will my sequencing library have covered all the genomic copies used as template? Is there a way to calculate how many I have covered? Is there a library preparation step to control this parameter?
Thank you very much!