Sequence information provides reliable data for microbial genotyping applications. However, standard methods used to assess discriminatory regions of microbial genes can be time-consuming, may require species-specific probes or gel electrophoresis, or are susceptible to the presence of unknown mutations (e.g., hybridization). Pyrosequencing is a rapid and accurate alternative method. Because this technology sequences by synthesizing new copies of the DNA template, the results provide unambiguous information, allowing users to check the sequence surrounding the variable site and ensure that the correct DNA region has been analyzed.
Advantages of Pyrosequencing in microbial research
Pyrosequencing is a versatile tool that facilitates genotyping and quantitative examination of sequence variation. Advantages of Pyrosequencing analysis for microbiology applications include:
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- Rapid and reliable high-throughput screening
- Accurate identification of unknown mutations
- Sequence context provides built-in quality control of the assay
- No need for expensive labels and dyes
- Significant time and cost savings
Identify multiple species in one run and with one primer set
Unlike hybridization techniques, Pyrosequencing allows the identification of a large number of species using a single conserved sequencing primer (see figure Pyrosequencing-based bacterial identification
). Consequently, DNA extracted from multiple microbe species can be sequenced in the same Pyrosequencing run. PyroMark IdentiFire SW 1.0 facilitates the compilation of a local sequence database against which imported Pyrosequencing output are rapidly aligned. The raw data, matched hits, and percent concordance of each hit are presented in detailed identification reports. These reliable data are provided within short timeframes. In less than one hour, Pyrosequencing reads a discriminatory stretch of DNA of up to 96 samples in parallel (see figure Time savings afforded by Pyrosequencing
). Depending on assay design, the sequence can be used to discriminate microbial species, types and strains, or detect genetic mutations that confer resistance to antibiotics and antiviral drugs.
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Tracking drug resistance development
Multiple samples can be concurrently assayed for common drug resistance mutations (see figure Analysis of
antibacterial resistance in helicobacter pylori
). Since Pyrosequencing reports the actual sequence of the locus, a single assay can detect a range of possible mutations, including new and unexpected mutations. Instrumentation and optimization of assays make Pyrosequencing technology routinely capable of detecting sequence variants present at frequencies as low as 5%, and potentially lower for some targets. Furthermore, heterogenic variation among several gene copies, which accounts for different resistance patterns, is reliably quantified. Visualizing the mutation in the context of the DNA sequence permits the user to verify the analysis. Coupled with the short time to obtain results and the ease of use, this analysis flexibility and detection sensitivity make Pyrosequencing an informative complement or even alternative to culture-based and hybridization-based resistance screening.
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A growing number of species-specific assays
While broad-range identification strategies using signature sequences in well-characterized hypervariable regions of microbial genes have been described, there are a growing number of assays in the literature that specifically target commonly studied pathogenic species and strains of bacteria, viruses, and fungi.