Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays

Bioinformatics, Aug 2011

Motivation: Many current studies of complex microbial communities rely on the isolation of community genomic DNA, amplification of 16S ribosomal RNA genes (rDNA) and subsequent examination of community structure through interrogation of the amplified 16S rDNA pool by high-throughput sequencing, phylogenetic microarrays or quantitative PCR. Results: Here we describe the development of a mathematical model aimed to simulate multitemplate amplification of 16S ribosomal DNA sample and subsequent detection of these amplified 16S rDNA species by phylogenetic microarray. Using parameters estimated from the experimental results obtained in the analysis of intestinal microbial communities with Microbiota Array, we show that both species detection and the accuracy of species abundance estimates depended heavily on the number of PCR cycles used to amplify 16S rDNA. Both parameters initially improved with each additional PCR cycle and reached optimum between 15 and 20 cycles of amplification. The use of more than 20 cycles of PCR amplification and/or more than 50 ng of starting genomic DNA template was, however, detrimental to both the fraction of detected community members and the accuracy of abundance estimates. Overall, the outcomes of the model simulations matched well available experimental data. Our simulations also showed that species detection and the accuracy of abundance measurements correlated positively with the higher sample-wide PCR amplification rate, lower template-to-template PCR bias and lower number of species in the interrogated community. The developed model can be easily modified to simulate other multitemplate DNA mixtures as well as other microarray designs and PCR amplification protocols. Contact: oleg.paliy{at}wright.edu Supplementary information: Supplementary data are available at Bioinformatics online.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://bioinformatics.oxfordjournals.org/content/27/15/2134.full.pdf

Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays

Oleg Paliy 1 Brent D. Foy 0 Associate Editor: Jonathan Wren 0 Department of Physics, Wright State University , Dayton, OH 45435, USA 1 Department of Biochemistry and Molecular Biology Motivation: Many current studies of complex microbial communities rely on the isolation of community genomic DNA, amplification of 16S ribosomal RNA genes (rDNA) and subsequent examination of community structure through interrogation of the amplified 16S rDNA pool by high-throughput sequencing, phylogenetic microarrays or quantitative PCR. Results: Here we describe the development of a mathematical model aimed to simulate multitemplate amplification of 16S ribosomal DNA sample and subsequent detection of these amplified 16S rDNA species by phylogenetic microarray. Using parameters estimated from the experimental results obtained in the analysis of intestinal microbial communities with Microbiota Array, we show that both species detection and the accuracy of species abundance estimates depended heavily on the number of PCR cycles used to amplify 16S rDNA. Both parameters initially improved with each additional PCR cycle and reached optimum between 15 and 20 cycles of amplification. The use of more than 20 cycles of PCR amplification and/or more than 50 ng of starting genomic DNA template was, however, detrimental to both the fraction of detected community members and the accuracy of abundance estimates. Overall, the outcomes of the model simulations matched well available experimental data. Our simulations also showed that species detection and the accuracy of abundance measurements correlated positively with the higher sample-wide PCR amplification rate, lower template-to-template PCR bias and lower number of species in the interrogated community. The developed model can be easily modified to simulate other multitemplate DNA mixtures as well as other microarray designs and PCR amplification protocols. Contact: Supplementary information: Supplementary data are available at Bioinformatics online. 1 INTRODUCTION Owing to the development and refinement of novel DNA and RNA interrogation technologies, there is a surge of studies in the current literature exploring the populational structure and function of various complex microbial communities (Brodie et al., 2007; Gao et al., 2007; Huber et al., 2007). The ability to isolate and subsequently examine total community DNA and RNA without any need to culture individual microbial species and cells allows analysis of systems that would otherwise be difficult to profile and examine including microbiota of human intestine and other epithelial surfaces and the microbes of soils and ocean waters (Eckburg et al., 2005; Gao et al., 2007; Huber et al., 2007; Kent and Triplett, 2002). Gene coding for the small ribosomal subunit RNA molecule (16S rRNA in prokaryotes and 18S rRNA in eukaryotes) has been used in the vast majority of such studies due to its ubiquitous presence in all organisms and because of the conservation of its nucleotide sequence (Cannone et al., 2002). In a typical experimental design to profile microbial community structure, total genomic DNA (gDNA) isolated from a sample of interest is subjected to rounds of 16S rRNA gene (rDNA) specific amplification in polymerase chain reaction (PCR) using two universal primers complementary to the beginning and the end of prokaryotic 16S rRNA molecule (Frank et al., 2008). The amplified DNA is then interrogated by a detection method of choice such as DNA sequencing or microarray analysis. Because on average 16S rRNA gene constitutes only 0.25% of the total genomic DNA (see below), selective 16S rDNA amplification is crucial to increase the sensitivity of detection (Paliy et al., 2009) and to obtain good measures of bacterial presence and relative abundance in the community samples. However, the optimal use of such PCR amplification in relation to microarray and DNA sequencing detection have not been yet fully explored. A number of studies have been published, though, examining the thermodynamic behavior of DNA molecules during DNA amplification, and the biases that can be observed during many rounds of PCR amplification (Kanagawa, 2003; Kurata et al., 2004; Polz and Cavanaugh, 1998). Because most microbial communities consist of a large number of different microbial species with varied 16S rRNA gene sequences, any PCR amplification of community DNA is multitemplate. PCR amplification of such gDNA has been shown to introduce a deviation of the post-amplification fractions from the initial ratios of DNA molecules (termed PCR bias) due to unequal amplification of different DNA molecules during PCR (Polz and Cavanaugh, 1998). Several mechanisms of this effect have been described that include (i) unequal denaturation of templates based on GC content of DNA sequences; (ii) higher binding efficiency of GC-rich variants of degenerate amplification primers to the template at the same annealing temperature; and (iii) competitive re-annealin (...truncated)


This is a preview of a remote PDF: https://bioinformatics.oxfordjournals.org/content/27/15/2134.full.pdf

Oleg Paliy, Brent D. Foy. Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays, Bioinformatics, 2011, pp. 2134-2140, 27/15, DOI: 10.1093/bioinformatics/btr326