Provides a basic understanding of 16S RNA sequencing which can associate to its advance involvement in metagenomics

1. 16S rDNA Sequence Analysis

2. • What are the characteristics of 16S rRNA that
make it
• useful as an analyte for bacterial identification?
• • Ubiquitous
• • Highly Conserved Molecule
• • Contains variable and hyper-variable regions
of sequence
• • Extensively studied and represented in
database

4. STRUCTURES
• •Primary Structure
• RNA sequence
• Secondary Structures
• Formation of double
• stranded regions (helices)
• Tertiary Structures
• Folding of 1o and 2o structures
• Quaternary Structures
• Interactions with other
• molecules (e.g. RNAs, proteins)

5. Small Subunit rRNA
16S in Eubacteria
18S in Eukaryotes
E.coli 16S rRNA Molecule
Typically used for comparison

6. Why Mycobacteria?
• • Slow growing and fastidious
• • Phenotypic identification can take 2 to 8 weeks
• • Cultural data: Growth rate, pigmentation and
morphology
• • Accu-probe testing: Can test for M.tuberculosis, MAC,
• M.kansasii and M.gordonae only.
• ••
• Biochemical tests: Tween 80 hydrolysis, arylsulfatase etc.
• ••
• HPLC, GLC, TLC: for lipid wall analysis

8. Precious Time is Wasted
• Patients might receive inappropriate therapy.
• Point mutations can confer antimicrobial
resistance
• Atypical Mycobacteria respond differently to
standard
• antimicrobials

9. Other Gene Targets
•Hsp 65 : 65 kDa gene which is also highly
conserved
• Highly specific for Mycobacteria
•recA gene

12. Pilot Study
• • 22 different cultures of microorganissms
subjected to
• 16S rDNA sequence analysis
• •
• • Results compared to phenotypic data
• •
• • Includes several Mycobacterium cultures

13. Methods Outline
• •Isolate bacterial DNA
• •Amplify 16S rRNA gene
• •Sequence a portion of 16S rRNA gene (Region
1)
• •Compare sequence obtained with GenBank to
find “Match”

14. Sequence Analysis

15. Compare to GenBank
• •Gen Bank is a freely available web-based
database
• of 16S rDNA sequences
• •Contains bacteria, fungi and other
microrganisms
• •BLAST “match” was done

16. Advantages
• •Prevents misidentification by phenotypic analysis
• •Early diagnosis
• •Validated by numerous studies
• •Not subject to false-negative results
• •Can be matched against GenBank, RIDOM and the
ribosomal database library
• •Identification of unusual isolates

17. Drawbacks
• •Ambiguous data in databases: base errors,
incomplete sequences
• •Difficult to differentiate some organisms like
• M. chelonae and M. abcessus

18. Clinical Relevance
• •Early detection of microrganisms
• •Needs very little substrate to work with
• •Better targeting of antimicrobials
• •Used successfully by Sacchi et al in the recent
bioterrorism outbreak for early detection of B.
anthracis
• •Incorporated into routine diagnostics at a number of
centers