I plan to do nifH targeted NGS (most likely paired end reads with MiSeq) to study free living nitrogen microbial communities in different tillage management system soil samples. I see as much as possible species coverage.I am looking for some guidance to help me to decide how many reads or enough deep sequencing I need per sample?
Hi Manoj,
I'm assuming you are doing amplicon sequencing.
If so I would target 50,000 reads per sample as a reasonable starting point. This should give you about 4 log dynamic range in detecting higher and lower abundance species.
Please note, the limitation of the dynamic range can easily be due to number of input genomes into the amplification rather than the sequencing depth. If your only putting in 1000 genomes to start, there is no need to go above 10,000 reads. I use 5MB/genome as a rule of thumb and then use input DNA concentration and volume to estimate genomes going in. Targeting reads at 10X number of genomes going is not perfect, but it does cover some of the potential variation in amplification efficiency and Poisson distribution effects.
Hope this helps,
Frank
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