Agriculture Reference
In-Depth Information
Microbial Communities in Soil
lyse cells
extract DNA
Purified
DNA
Gene
Abundance
Q-PCR
PCR
tRFLP
Profile
Shotgun
Metagenomes
Clone
Libraries
DNA Sequences
Taxonomic & Genetic
Composition
Diversity
Richness & Eveness
Figure 6.1 . An overview of molecular approaches presented in this chapter that have been
used to assess the composition and diversity of microbial communities in KBS LTER soils.
The polymerase chain reaction (PCR) is used commonly to obtain sufficient quantities of
a target gene for subsequent analysis. Quantitative PCR (Q-PCR) is used to estimate gene
abundance, while terminal restriction fragment length polymorphism (tRFLP) is a finger-
printing method that provides an overview of gene diversity.
The 16S and 18S rRNA-encoding genes contain regions of strict sequence con-
servation that are valuable for the design of universal primers to amplify the genes
via PCR. These conserved regions are interspersed with regions of sequence vari-
ability that are useful for comparative analysis. A major advantage of targeting the
SSU rRNA gene for surveys of diverse communities is the opportunity for compari-
son with the approximately 1.5 million other SSU rRNA gene sequences in curated
public databases (Pruesse et al. 2007, Cole et al. 2009). Amplified 16S genes can
either be cloned into plasmid vectors and sequenced individually, or sequenced
directly with massively parallel “next-generation” sequencing. Because of their
high capacity, next-generation technologies offer the potential for considerable
in-depth exploration of diversity (Sogin et al. 2006). The 16S gene sequences can
be compared at several thresholds of sequence similarity, providing insight into the
composition of microbial communities at different taxonomic levels.
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