Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.
Total time
~4–6 hours per sample batch (PCR through sequencing run completion)
Steps
1
Amplify microbial template DNA via PCR
Perform PCR amplification of target microbial genomic regions to generate biotinylated amplicons suitable for downstream pyrosequencing analysis.
▶ 02:40
2
Prepare samples using vacuum workstation
Apply vacuum workstation protocol to process amplified PCR products for immobilization.
▶ 04:01
3
Immobilize biotinylated PCR products onto beads
Bind biotinylated amplicons to streptavidin-coated magnetic beads using established immobilization protocol.
▶ 04:05
4
Denature DNA and add sequencing primer
Denature double-stranded DNA templates and anneal sequencing-specific primer for single-strand preparation.
▶ 05:06
5
Anneal sequencing primer to DNA strands
Allow sequencing primer to hybridize to complementary single-stranded DNA template.
▶ 07:30
6
Configure Pyromark Q24 instrument settings
Set up and configure the Pyromark Q24 pyrosequencer with assay-specific parameters and reaction conditions.
▶ 07:55
7
Load samples and prepare for sequencing run
Load prepared samples into the instrument cartridge and perform final quality checks before initiating the pyrosequencing run.
▶ 07:58
8
Initiate automated pyrosequencing run
Start the automated pyrosequencing reaction; enzyme and reagent dispensing proceeds according to programmed protocol.
▶ 08:58
9
Complete sequencing run and collect data
Allow run to complete; instrument automatically detects light signals and generates raw sequence data for analysis.
▶ 09:46
10
Analyze results for species and resistance identification
Interpret pyrograms and sequence data to identify bacterial species and detect antibiotic resistance-conferring mutations.
▶ 10:03