Abstract
The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.
Highlights
Knowing the relative abundance of individual taxa reveals important information about any ecological community, including microbial communities
We developed host-associated microbe PCR (hamPCR), a simple and robust method to quantitatively co-amplify one or more microbial marker genes along with an unrelated host gene, allowing accurate determination of microbial load and microbial community composition from a single sequencing library
We developed a method to predictably optimize the amount of sequencing effort devoted to microbe vs. host, without losing information about the original microbe to host ratio (Figure 3)
Summary
Knowing the relative abundance of individual taxa reveals important information about any ecological community, including microbial communities. Researchers combine amplicon sequencing with an additional orthogonal method These include supplementary shallow WMS (Regalado et al, 2020), quantitative PCR (qPCR) or digital PCR of host and/or microbial genes (Anderson and McDowell, 2015; Barlow et al, 2020; Ellegaard et al, 2020; Guo et al, 2019; Jian et al, 2020; Karasov et al, 2019; Nadkarni et al, 2002), adding sequenceable “spike-ins” calibrated based on sample volume (Lin et al, 2019), mass (Stämmler et al, 2016), or qPCR-determined host DNA content (Guo et al, 2019), counting colony forming units (CFU)(Chen et al, 2020; Niu et al., 2017), and flow cytometry (Props et al, 2016; Vandeputte et al, 2017). Because of the practical simplicity and flexibility of hamPCR, it has the potential to supplant traditional microbial amplicon sequencing in host-associated microbiomes
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