4.2.2. Spike-ins
Introducing an internal standard (also called a spike-in) can be a useful tool toward achieving more quantitative amplicon data analyses. Spike-ins can be introduced in the form of microbial cells \cite{St_mmler_2016} or as selected DNA sequences \cite{Tkacz_2018,Hardwick2018,Wang2021}. The spike should be uniquely detectable as a non-member of the existing microbial community, and should not be introduced in concentrations that would shift the sequencing effort towards it. Additionally, the timing of the addition will determine the type of information retrieved. While adding the spike after extraction can provide good estimates of amplification and/or sequencing biases, it does not take extraction efficiency into account \cite{Hardwick2018,St_mmler_2016}. A recent amplicon sequencing study applied a synthetic DNA spike of known concentration to faecal samples prior to extraction. They combined this with qPCR quantification to calculate the number of gene copies after accounting for the extraction yield. The ratio of each OTU against the initial concentration of 16S rRNA genes was used to calculate more accurate abundance levels of each OTU after taking extraction efficiency into account \citep{Zemb2020}. If performed in a comparable manner, spike-ins represent a promising tool to determine abundances of taxa more quantitatively via sequencing in future soil studies.