Thus, for whole-genome sequencing, the more specific technique of fluorescence quantitation of DNA is greatly preferred. The
fluorescent dyes used for this purpose generally work by intercalation between the base pairs of double-stranded DNA. Because the
dye therefore cannot bind to either ssDNA or RNA, the fluorescence emitted is specific to dsDNA. This makes the resulting calculated DNA concentration far more accurate for the purposes
of WGS.
3.3.2 Purity
However, despite its deficiencies for accurate quantitation, UV
spectrophotometry is still a valuable part of DNA QC. The presence of contaminating molecules in a DNA sample can dramatically
decrease the quality of the results obtained through WGS. The
purity of a DNA sample is typically assessed using ratios of the
absorbance of ultraviolet light at three wavelengths: 230, 260,
and 280 nm (A 230 , A 260 , and A 280 , respectively).
An A 260 /A 280 ratio of %1.8 represents a highly pure sample of
DNA. On the other hand, a sample with a significant quantity of
RNA will have an A 260 /A 280 ratio of %2.0. A sample with an A 260 /
A 280 ratio below 1.8 is likely contaminated with proteins.
It is important to fully remove any chemicals introduced in the
DNA extraction process as well as any unwanted cellular debris.
The presence of these contaminants can have an adverse effect on
WGS. Because many of these potential contaminants absorb ultraviolet light at %230 nm, examination of the A 260 /A 230 ratio can
provide some indication of a sample’s purity with respect to such
contaminants. Generally, an A 260 /A 230 ratio greater than 2.0 indicates a high-purity sample, whereas a value below 1.5 indicates
significant contamination with such chemicals.
3.3.3 Fragment Length
As mentioned in Subheading 3.1, if the DNA sample is intended for
use with a long-read sequencing platform, the individual strands of
DNA should be as long as possible. This allows the generation of
individual reads of the greatest length, enabling the better resolution of complex or repetitive regions. Qualitatively, a portion of the
DNA solution can be analyzed via electrophoresis using a
low-concentration (%0.8%) agarose gel. The degree of smearing
seen on the gel corresponds to distribution of fragment sizes of the
DNA. Ideally, there should be a large bright band toward the top of
the gel; this indicates that the majority of DNA is in fragments so
large that they cannot easily migrate down the gel. A high-quality
extraction yielding high-molecular weight DNA should have a
minimal smear under the main bright band. A smaller top band
and a more prominent smear indicate that the DNA was sheared
during extraction, which could negatively affect the quality of
sequencing data generated. If this is the case, the user may prefer
instead to re-extract the DNA from the organism, taking greater
care during the extraction process to maintain the integrity of the
DNA strands.
Mycobacterial ID and Characterization Using WGS
409
fluorescent dyes used for this purpose generally work by intercalation between the base pairs of double-stranded DNA. Because the
dye therefore cannot bind to either ssDNA or RNA, the fluorescence emitted is specific to dsDNA. This makes the resulting calculated DNA concentration far more accurate for the purposes
of WGS.
3.3.2 Purity
However, despite its deficiencies for accurate quantitation, UV
spectrophotometry is still a valuable part of DNA QC. The presence of contaminating molecules in a DNA sample can dramatically
decrease the quality of the results obtained through WGS. The
purity of a DNA sample is typically assessed using ratios of the
absorbance of ultraviolet light at three wavelengths: 230, 260,
and 280 nm (A 230 , A 260 , and A 280 , respectively).
An A 260 /A 280 ratio of %1.8 represents a highly pure sample of
DNA. On the other hand, a sample with a significant quantity of
RNA will have an A 260 /A 280 ratio of %2.0. A sample with an A 260 /
A 280 ratio below 1.8 is likely contaminated with proteins.
It is important to fully remove any chemicals introduced in the
DNA extraction process as well as any unwanted cellular debris.
The presence of these contaminants can have an adverse effect on
WGS. Because many of these potential contaminants absorb ultraviolet light at %230 nm, examination of the A 260 /A 230 ratio can
provide some indication of a sample’s purity with respect to such
contaminants. Generally, an A 260 /A 230 ratio greater than 2.0 indicates a high-purity sample, whereas a value below 1.5 indicates
significant contamination with such chemicals.
3.3.3 Fragment Length
As mentioned in Subheading 3.1, if the DNA sample is intended for
use with a long-read sequencing platform, the individual strands of
DNA should be as long as possible. This allows the generation of
individual reads of the greatest length, enabling the better resolution of complex or repetitive regions. Qualitatively, a portion of the
DNA solution can be analyzed via electrophoresis using a
low-concentration (%0.8%) agarose gel. The degree of smearing
seen on the gel corresponds to distribution of fragment sizes of the
DNA. Ideally, there should be a large bright band toward the top of
the gel; this indicates that the majority of DNA is in fragments so
large that they cannot easily migrate down the gel. A high-quality
extraction yielding high-molecular weight DNA should have a
minimal smear under the main bright band. A smaller top band
and a more prominent smear indicate that the DNA was sheared
during extraction, which could negatively affect the quality of
sequencing data generated. If this is the case, the user may prefer
instead to re-extract the DNA from the organism, taking greater
care during the extraction process to maintain the integrity of the
DNA strands.
Mycobacterial ID and Characterization Using WGS
409