Next-Generation Sequencing
Sanger Sequencing
Has been the major method for last 30 years
Revolutionised molecular biology
Main change is number of samples sequenced has increased
HOWEVER length of reads per run has only improved slightly over the years.
Limitations
Maximum read length usually <1000bp
High cost per base
Long experimental set up times
High DNA concentrations needed
Some regions cannot be sequenced (G+C content
rich – due to annealing and hybridising properties
Sequencing by Synthesis…
Single DNA molecules replicated
Replicated DNA made single stranded
Replicates (clonal amplifications) are fixed to a specific location
Sequencing occurs using a DNA polymerase based method
Sanger sequencing does not read the sequence as is it synthesised
454 Sequencing by 454 (Roche)
1. Ds target DNA (genomic or
more specific) broken up into
400-600bp fragments
- DNA shearing can be done
randomly using a nebulizer
then “polish” the DNA (digest
with S1 nuclease) to achieve
blunt ends
2. Two 44bp adaptors (A + B) are
ligated to each end of the
fragments
Adaptors A + B
Sanger Sequencing
Has been the major method for last 30 years
Revolutionised molecular biology
Main change is number of samples sequenced has increased
HOWEVER length of reads per run has only improved slightly over the years.
Limitations
Maximum read length usually <1000bp
High cost per base
Long experimental set up times
High DNA concentrations needed
Some regions cannot be sequenced (G+C content
rich – due to annealing and hybridising properties
Sequencing by Synthesis…
Single DNA molecules replicated
Replicated DNA made single stranded
Replicates (clonal amplifications) are fixed to a specific location
Sequencing occurs using a DNA polymerase based method
Sanger sequencing does not read the sequence as is it synthesised
454 Sequencing by 454 (Roche)
1. Ds target DNA (genomic or
more specific) broken up into
400-600bp fragments
- DNA shearing can be done
randomly using a nebulizer
then “polish” the DNA (digest
with S1 nuclease) to achieve
blunt ends
2. Two 44bp adaptors (A + B) are
ligated to each end of the
fragments
Adaptors A + B
