Internal Genomic DNA Standard for Quantitative Metagenome Analysis
Suspend genomic DNA in a volume of nuclease-free water to produce a stock concentration of 0.1 μg/μL.
Incubate rehydrated DNA overnight at 4°C while rocking, and then incubate for 1 h at 65°C.
Prepare working solution by adding 1 μL of the stock solution to 99 μL of nuclease-free water to produce a final concentration of 1 ng/μL.
Check the DNA concentration of stocks fluorometrically using Quant-iT™ PicoGreen® dsDNA Assay Kit Genomic DNA can be stored at −20 °C.
Just prior to sample DNA extraction, add enough standard DNA to each sample to reach ~0.1-1.0% of expected total reads.
Following sequencing, quantify the number of genomic standard reads (steps 8 and 9).
Using a bit score cutoff of 50, identify standard reads by BLASTn homology search aganst the internal standard genome.
Using the results from step 8 and a bit score cutoff of 40, perform a BLASTx against the RefSeq Protein database to identify all protein encoding reads derived from the internal standard genome.
Quantify recovered standard DNA reads and remove from dataset.
Calculate the number of molecules of internal standard recovered from sequencing: Sr=SS/SPSr = number of molecules of internal standard genome recovered from sequencingSS = number of protein encoding internal standard reads in the sequence librarySP = number of protein encoding genes in the internal standard reference genome. 
Calculate the community gene pool size: Pg=Ps*(Sa/Sr)Pg = total number of protein encoding genes in the samplePs = number of protein encoding sequences in the metagenome librarySa = number of molecules of internal standard genome added to the sampleSr = number of molecules of internal standard genome recovered from sequencing. 
Calculate individual gene abundances for genes of interest: Ga=Gs*(Pg/Ps)Ga = number of molecules of any particular gene category in the sample.
This can then be divided by the mass or volume of sample collected to calculate the gene abundance per volume or weightGs = number of genes of interest in the sequence libraryPg = total number of protein encoding genes in the samplePs = number of protein encoding sequences in the metagenome library
