Oxford Nanopore
8 devices do not rely on DNA replication chemistry at all. While
adapters are used to help ensure proper orientation of DNA molecules as they
interact with the nanopore, polymerase, dNTPs, and sequencing primers are needed
for sequencing (Fig. 6). This makes the Oxford Nanopore technique the first
technique since Maxam-Gilbert sequencing to not require primers and to not utilize
DNA replication or ligation chemistry (at least in the sequencing phase). DNA
molecules are currently sequenced using 2D and 1D
2 strategies (see Fig. 6). The
advantage of the 2D strategy is that the template and complementary strand are
sequenced in a single read. The advantage of the 1D
2 strategy is that if the template
strand breaks during sequencing, it will not prevent sequencing of the complementary strand. The error rate of Oxford Nanopore reads is currently higher than other
NGS methods, but considerable improvements have been made (Jain et al. 2016).
Oxford Nanopore read lengths are unparalleled (Table 1).
Both PacBio and Oxford Nanopore technologies have expanded their markets
considerably over the last 2 years. PacBio is in the driver’s seat at the moment in
terms of sales, but there are several indicators that Oxford Nanopore may be close to
overthrowing PacBio as the leader in long-read sequencing technology (Greenleaf
and Sidow 2014; Heather and Chain 2016).
3.11 Contig
A contig (short for contiguous sequence) is a set of partially overlapping sequence
reads ostensibly representing a continuous piece of genomic DNA (Fig. 7). The term
contig is also used to describe a set of partially overlapping BACs or other genomic
DNA clones.
3.12 Consensus Sequence
A DNA sequence derived from a contig where the most common base found at a
given position along aligned sequence reads is used to represent that position
(Fig. 7).
8 As their name suggests, nanopores are simply extremely small pores. They can be derived from
naturally existing protein-based membrane pores (e.g., α-hemolysin and Mycobacterium smegmatis
porin A) or simply etched into silicon or other substrates (solid-state nanopores). Numerous
laboratories around the world have been working on using biological and solid-state nanopores to
characterize a variety of different molecules including DNA, RNA, proteins, enantiomers, drugs,
and polymers (Haque et al. 2013). To date, Oxford Nanopore is the only group to produce
commercially available nanopore-based DNA sequencing instruments (Heather and Chain 2016;
Jain et al. 2016).
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D. G. Peterson and M. Arick
8 devices do not rely on DNA replication chemistry at all. While
adapters are used to help ensure proper orientation of DNA molecules as they
interact with the nanopore, polymerase, dNTPs, and sequencing primers are needed
for sequencing (Fig. 6). This makes the Oxford Nanopore technique the first
technique since Maxam-Gilbert sequencing to not require primers and to not utilize
DNA replication or ligation chemistry (at least in the sequencing phase). DNA
molecules are currently sequenced using 2D and 1D
2 strategies (see Fig. 6). The
advantage of the 2D strategy is that the template and complementary strand are
sequenced in a single read. The advantage of the 1D
2 strategy is that if the template
strand breaks during sequencing, it will not prevent sequencing of the complementary strand. The error rate of Oxford Nanopore reads is currently higher than other
NGS methods, but considerable improvements have been made (Jain et al. 2016).
Oxford Nanopore read lengths are unparalleled (Table 1).
Both PacBio and Oxford Nanopore technologies have expanded their markets
considerably over the last 2 years. PacBio is in the driver’s seat at the moment in
terms of sales, but there are several indicators that Oxford Nanopore may be close to
overthrowing PacBio as the leader in long-read sequencing technology (Greenleaf
and Sidow 2014; Heather and Chain 2016).
3.11 Contig
A contig (short for contiguous sequence) is a set of partially overlapping sequence
reads ostensibly representing a continuous piece of genomic DNA (Fig. 7). The term
contig is also used to describe a set of partially overlapping BACs or other genomic
DNA clones.
3.12 Consensus Sequence
A DNA sequence derived from a contig where the most common base found at a
given position along aligned sequence reads is used to represent that position
(Fig. 7).
8 As their name suggests, nanopores are simply extremely small pores. They can be derived from
naturally existing protein-based membrane pores (e.g., α-hemolysin and Mycobacterium smegmatis
porin A) or simply etched into silicon or other substrates (solid-state nanopores). Numerous
laboratories around the world have been working on using biological and solid-state nanopores to
characterize a variety of different molecules including DNA, RNA, proteins, enantiomers, drugs,
and polymers (Haque et al. 2013). To date, Oxford Nanopore is the only group to produce
commercially available nanopore-based DNA sequencing instruments (Heather and Chain 2016;
Jain et al. 2016).
136
D. G. Peterson and M. Arick