LT16 Next-Gen Sequencing

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