16S rRNA
Muna Salah
16S rRNA gene sequencing
• Highly conserved
regions
– Identical in all bacteria
– Single PCR primer pair
can amplify 16S rRNA
genes from diverse
bacteria
• Highly variable regions
– Conserved within
species
– Divergent
g
between
species
Image from Alimetrics.net
16S rRNA Sequences
• The
h genes coding
di for
f 16S rRNA, 23S rRNA, and
d
5S rRNA are essential for existence of each
bacterial cell due to its involvement in protein
synthesis.
• Their sequences are generally highly
conserved amongg bacteria.
• Some variable regions are present within
these sequences.
sequences
• 16S rRNA is conserved at the strain level.
Primer forward (Upper; 5’)
Samp_A
Samp_B
Samp_C
Samp_D
Samp_E
Samp_F
Samp_G
Primer reverse (Lower; 3’)
5’ATCGCTCGATCGTAAATCGCGTTAGCGGTAGCTATACGATTTATCGATCGTATACGTAGCGTAGCGTAATTTTAAAACCGT
5’.......................G................A........................................
5’............................C............................G.......................
5’.......................G.........................................................
5’.......................................AC..........T.............................
5’...................................T.............................................
5’...................................T.........._..........C.......................
Sequence
BLAST
3’
3’
3’
3’
3’
3’
3’
Assay Workflow
Culture,
Biopsy
Nucleic acid
extraction
Amplification
of the
TARGET
GE
region (PCR)
DNA
sequence
Result ! - Identification at
Species / Subspecies level
Sequence alignment
(BLAST – NCBI)
General 16S rRNA sequencing strategy
• IIsolate
l t DNA
• Amplify 16S rRNA genes using PCR and primers
recognizing
i i conserved
d regions
i
• Perform NGS
– usually Roche 454
• Use sequence data to identify types and
abundances of bacterial “species”
• Measure community diversity (alpha)
• Compare diversity between communities,
locations treatments,
locations,
treatments etc.
etc (beta)
Why use 16s rRNA gene sequencing
The 16s rRNA gene sequence show a high degree of conservation among
species This is assumed to result from the importance of the 16S rRNA as a
species.
critical component of cell function.
Few other genes are as highly conserved as the 16S rRNA gene.
gene Although the
absolute rate of change in the 16S rRNA gene sequence is not known, it does
mark evolutionary distance and relatedness of organisms
16s rRNA is present in almost all bacteria, often existing as a multigene
family, or operons.
The 16s rRNA gene (1,500
(1 500 bp) is large enough for informatics purposes.
purposes
16s rRNA gene sequencing studies as opposed to the more cumbersome
a pu at o s involving
o
g DNA-DNA hybridization
yb d at o investigations.
est gat o s
manipulations
16s rRNA gene of various bacteria are extensively studied and is present in
many databases
Generating Phylogenetic Trees and
Comparing Sequences
• Comparisons are commonly shown as
phylogenetic trees and linear alignments.
• A phylogenetic tree is a tree‐structured graph
used
d in
i computational
i
l biology
bi l
to visualize
i li the
h
result of a hierarchical clustering calculation.
• The result of a clustering is presented either as
the distance(dissimilarity) or the similarity
between the clustered rows or columns
depending on the selected distance measure
measure.
Methods commonly used for generating
phylogenetic trees

NJ method (Neighboor
(Neighboor-joining)
joining),

UPGMA method (Unweighted pair group method with
arithmetic averages)

WPGMA method ((Weighted
g
p
pair g
group
p method with
arithmetic averages)
APPLICATIONS OF BACTERIAL IDENTIFICATION
USING 16S rRNA SEQUENCING
Identifying
Id
if i unidentified
id ifi d b
bacteria
i or iisolates
l
with
i h ambiguous
bi
profiles.
•
One of the most attractive potential uses of 16S rRNA gene sequence
informatics is to provide genus and species identification for isolates that do
not fit any recognized biochemical profiles, for strains generating only a “low
likelihood” or “acceptable” identification according to commercial systems,
systems
or for taxa that are rarely associated with human infectious diseases.
•
The cumulative results from a limited number of studies to date suggest that
16S rRNA gene sequencing provides genus identification in most cases
(>90%) but less so with regard to species (65 to 83%), with from 1 to 14% of
g
the isolates remainingg unidentified after testing.
Conclusion
•
The traditional identification of bacteria on the basis of phenotypic
characteristics is generally not as accurate as identification based on
genotypic methods.
•
Comparison of the bacterial 16S rRNA gene sequence has emerged as a
preferred genetic
p
g
technique.
q
16S rRNA ggene sequence
q
analysis
y can better
identify poorly described, rarely isolated, or phenotypically aberrant strains,
can be routinely used for identification of mycobacteria, and can lead to the
recognition of novel pathogens and noncultured bacteria.
bacteria
•
Problems remain in that the sequences in some databases are not accurate,
there is no consensus quantitative definition of genus or species based on 16S
rRNA gene sequence data,
Conclusion
•
The proliferation of species names based on minimal genetic and
phenotypic differences raises communication difficulties, and micro
heterogeneity in 16S rRNA gene sequence within a species is common
common.
•
p its accuracy,
y 16S rRNA ggene sequence
q
analysis
y lacks widespread
p
Despite
use beyond the large and reference laboratories because of technical and
cost considerations. Thus, a future challenge is to translate information
from 16S rRNA gene sequencing into convenient biochemical testing
schemes, making the accuracy of the genotypic identification available to
the smaller and routine clinical microbiology laboratories.