Long Read Sequencing
Long Read Sequencing
Long-read technologies are capable of sequencing single molecules of longer lengths, between 1,000 and >50,000 base pairs. Due to the single-molecule nature of the platforms, sequencing can be performed directly on genomic DNA with no need for PCR. This reduces bias commonly associated with amplification but means that large amounts of input material (a few micrograms) are often required. In addition, DNA extraction must be optimised to prevent damage (e.g. shearing with pipette usage) and remove contaminants which may inhibit library preparation and sequencing (e.g. bound polysaccharides, phenol, EDTA).
The volume of data produced per run is significantly less than an Illumina short read sequencer such as the NovaSeq 6000. However, the multi-kilobase reads available from long read platforms enable assembly of complete bacterial genomes and dramatically improve the contiguity of more complex genomes especially when large repetitive regions can benefit from the very long reads achievable. They also enable applications such as full-length transcript sequencing, reliable identification of structural variants and direct detection of epigenetic marks. A nice long-reads review article is shown here.
Pacbio and Nanopore sequencing can also be used in conjunction with short read data to improve existing assemblies.
The Sequel platform available from Pacific Biosciences (PacBio) is nolonger available for sequencing at the University of Exeter. Currently we are using Oxford Nanopore Technologies Promethion and Minion for our long read sequencing.