Comparison of molecular methodologies
| Technique | Standard culture | Fingerprinting methods | High throughput sequencing methods |
|---|---|---|---|
| Example | Culture on blood MacConkeys agar for Gram negative species | Temperature or Denaturing Gel electrophoresis (T/DGGE) | 454 pyrosequencing; ‘illumina’ etc |
| Brief description of how organisms are identified | Grown in a laboratory on specific media or with specific conditions; physical/chemical characteristics used to speciate | 16S rRNA isolated and amplified. Separation by sequence differences within amplicons using temperature or chemical gradient | Based on 16S rRNA extraction and sequencing, utilisinge extensive databases of 16S gene to identify species |
| Advantages | Lots of experience in technique; cheap; limited equipment needed; target key organisms | Greater depth of analysis than culture; cheap (for molecular technique); can be run against a ‘standard ladder’ | Very detailed information; complex communities can be examined; quick |
| Disadvantages | ∼90% species remain undetected; need to already know species of interest | Time consuming; separate steps needed to sequence amplicons and identify species; PCR bias | Costly; PCR bias; specialised equipment needed, available in few localities; enormous amounts of data generated require ‘handling’ and analysis |