the size of the input fragments. As such, careful handling of the
DNA to avoid shearing during library prep is particularly important. Indeed, using gentle extraction methods and cautious manipulation during library prep, mean read lengths can often reach into
the hundreds of kilobases, and the longest reads of 1–2 megabases
are not uncommon. However, as with PacBio sequencing, the
ONT method has an error rate much higher than sequence generated by the Illumina platforms. As such, the reads from PacBio or
ONT sequencing are often combined with Illumina reads to create
a hybrid assembly.
By combining both short and long reads, the hybrid assembled
genome can leverage the advantages of both sequencing technologies while complementarily minimizing the weaknesses of each.
Because they are able to span across most complex or repetitive
regions that are problematic to short-read-only assemblies, long
reads are typically used to first form a scaffold that is accurate at a
global structural level but is error-prone at the nucleotide level. The
short reads are then aligned to the scaffold, and their highly accurate sequence is used to correct (“polish”) the errors in the scaffold
[24]. This hybrid assembly method is able to generate highly
accurate finished genomes, e.g., with closed, circularized chromosomes and plasmids.
3.2 Nucleic Acid
Extraction
Techniques for nucleic acid extraction are described briefly in this
section. For additional information including detailed protocols,
please refer to other chapters.
DNA extraction encompasses the processes used to lyse bacterial cells (thus releasing the nucleic acid) and the isolation of the
DNA (thus concentrating and purifying it from the cellular debris).
For Gram-negative bacteria such as Escherichia coli, lysis can be
performed fairly simply and yields large quantities of high-quality
DNA. However, for many bacteria, the lysis process can present
difficulties. Gram-positive bacteria have a far thicker peptidoglycan
wall than do Gram-negative bacteria (10–20 layers vs. 1–2 layers).
This necessitates the use of harsher extraction methods, e.g.,
mechanical disruption such as beat beating, than are generally
required for Gram-negative species.
Extraction of DNA from mycobacteria poses similar problems
due to the presence of mycolic acids. Mycolic acids play a key role in
the structure of the unique mycobacterial outer membrane referred
to as the mycomembrane [25, 26]. Useful techniques can include
chemical lysis using cetyltrimethylammonium bromide (CTAB),
enzymatic extraction with lysozyme and proteinase K, mechanical
lysis such as beat beating or sonication, or various combinations
[27]. However, more vigorous extraction methods (particularly
bead beating) typically result in the shearing of DNA. Thus, the
selection of an appropriate extraction method becomes a balancing
act between obtaining sufficient yield and the quality of the
Mycobacterial ID and Characterization Using WGS
407
DNA to avoid shearing during library prep is particularly important. Indeed, using gentle extraction methods and cautious manipulation during library prep, mean read lengths can often reach into
the hundreds of kilobases, and the longest reads of 1–2 megabases
are not uncommon. However, as with PacBio sequencing, the
ONT method has an error rate much higher than sequence generated by the Illumina platforms. As such, the reads from PacBio or
ONT sequencing are often combined with Illumina reads to create
a hybrid assembly.
By combining both short and long reads, the hybrid assembled
genome can leverage the advantages of both sequencing technologies while complementarily minimizing the weaknesses of each.
Because they are able to span across most complex or repetitive
regions that are problematic to short-read-only assemblies, long
reads are typically used to first form a scaffold that is accurate at a
global structural level but is error-prone at the nucleotide level. The
short reads are then aligned to the scaffold, and their highly accurate sequence is used to correct (“polish”) the errors in the scaffold
[24]. This hybrid assembly method is able to generate highly
accurate finished genomes, e.g., with closed, circularized chromosomes and plasmids.
3.2 Nucleic Acid
Extraction
Techniques for nucleic acid extraction are described briefly in this
section. For additional information including detailed protocols,
please refer to other chapters.
DNA extraction encompasses the processes used to lyse bacterial cells (thus releasing the nucleic acid) and the isolation of the
DNA (thus concentrating and purifying it from the cellular debris).
For Gram-negative bacteria such as Escherichia coli, lysis can be
performed fairly simply and yields large quantities of high-quality
DNA. However, for many bacteria, the lysis process can present
difficulties. Gram-positive bacteria have a far thicker peptidoglycan
wall than do Gram-negative bacteria (10–20 layers vs. 1–2 layers).
This necessitates the use of harsher extraction methods, e.g.,
mechanical disruption such as beat beating, than are generally
required for Gram-negative species.
Extraction of DNA from mycobacteria poses similar problems
due to the presence of mycolic acids. Mycolic acids play a key role in
the structure of the unique mycobacterial outer membrane referred
to as the mycomembrane [25, 26]. Useful techniques can include
chemical lysis using cetyltrimethylammonium bromide (CTAB),
enzymatic extraction with lysozyme and proteinase K, mechanical
lysis such as beat beating or sonication, or various combinations
[27]. However, more vigorous extraction methods (particularly
bead beating) typically result in the shearing of DNA. Thus, the
selection of an appropriate extraction method becomes a balancing
act between obtaining sufficient yield and the quality of the
Mycobacterial ID and Characterization Using WGS
407