Biochemistry Questions Biochemistry Questions / Compare and contrast the advantages and disadvantages of second generation sequencing (e.g. Illumina) and third generation sequencing (e.g. PacBio and Oxford Nanopore). Which technology would you recommend for sequencing repetitive regions and identifying structural variants? Justify your answer.

Although second generation sequencing is currently the dominant technology, third generation sequencing will likely supersede it with longer read lengths, improved accuracy, and lower costs. Two third generation sequencing technologies are PacBio and Nanopore.

Longer read lengths are required when sequencing DNA with repeat regions. Although both PacBio (<50kb) and Nanopore (<2Mb) have longer sequence reads than Illumina (second generation sequencing, 150bp), Nanopore has the longer read length. These longer reads are advantageous when sequencing eukaryotic genomes with lengthy repeat regions, including the human genome. This makes it more unlikely that a discontinuous sequence will be produced after assembly, as longer repetitive regions can be processed and assembled without issue.

Both PacBio and Nanopore are capable of identifying structural variants. In PacBio sequencing, methylation is detected due to the longer fluorescence caused by the methylated nucleotide passing in to the polymerase. This is due to the nucleotide taking longer to pass through the polymerase. Nanopore sequencing produces a different electron signature as modified nucleotides pass through the pore protein, which can be processed as the data is captured.