extracted DNA. For short-read sequencing, the target fragmentation size during library prep is likely smaller than the size of the
sheared DNA strands, and so shearing during extraction likely plays
a minimal role. However, greater care must be exercised if the DNA
is to be used for long-read sequencing. Because the length of the
individual reads sequenced cannot exceed the length of the DNA
strands themselves, minimizing shearing is essential to generating
the longest reads possible.
Epperson et al. recently described an efficient protocol to
extract high-quality, largely intact DNA from M. tuberculosis and
diverse nontuberculous mycobacterial species [28], amenable to
scaling to 96 samples and avoiding noxious chemical such as phenol
and chloroform. The primary considerations in these cases are to
start with a single colony isolate, selected from growth on 7H10 or
7H11 agar, and then transfer for continued growth on 7H10 or
7H11 agar or 7H9 broth to a biomass appropriate for the sequencing technology intended. The starting material for the Epperson
protocol was single colony isolates selected and grown further in
either 7H9 broth or on 7H11 solid media for seven to ten days at
37
C for rapidly growing mycobacteria or two to four weeks at
37
C for slowly growing mycobacteria. A preference to increase
the number of inoculated cultures, as opposed to increasing the
growth time, was suggested to limit the amount of degraded DNA
resulting from older cells that could ultimately impact overall
sequence quality and read lengths [28]. Genomic DNA prepared
in this manner is also amenable to next-generation short-read
sequencing on platforms such as Illumina [29] or amplicon-based
Sanger sequencing.
3.3 Nucleic Acid
Quality
For the derived genomic sequence to be as reliable and as informative as possible, the sample of DNA to be sequenced should be of
the highest quality possible. The primary quality metrics that affect
sequencing are the concentration of DNA, its purity from contaminating chemicals and biomolecules, and potentially DNA fragment
length.
3.3.1 Concentration
For WGS to be successful, there must clearly be a sufficient quantity
of DNA in the sample to be sequenced. The two most common
methods for DNA quantification include spectrophotometry and
fluorescence quantitation. DNA spectrophotometry uses the absorbance of ultraviolet wavelengths (260 nm) to measure the concentration of nucleic acid in a sample. However, since DNA (both
single-stranded and double-stranded) and RNA absorb ultraviolet
light at this wavelength, contamination of the desired DNA with
RNA that was not degraded during the extraction will increase the
absorbance and thus the apparent concentration, leading to an
overestimation of the actual quantity of DNA in the sample. This
could lead to adding less DNA to the library prep process than is
optimal, resulting in a poor sequencing reaction.
408
Marco A. Riojas et al.
sheared DNA strands, and so shearing during extraction likely plays
a minimal role. However, greater care must be exercised if the DNA
is to be used for long-read sequencing. Because the length of the
individual reads sequenced cannot exceed the length of the DNA
strands themselves, minimizing shearing is essential to generating
the longest reads possible.
Epperson et al. recently described an efficient protocol to
extract high-quality, largely intact DNA from M. tuberculosis and
diverse nontuberculous mycobacterial species [28], amenable to
scaling to 96 samples and avoiding noxious chemical such as phenol
and chloroform. The primary considerations in these cases are to
start with a single colony isolate, selected from growth on 7H10 or
7H11 agar, and then transfer for continued growth on 7H10 or
7H11 agar or 7H9 broth to a biomass appropriate for the sequencing technology intended. The starting material for the Epperson
protocol was single colony isolates selected and grown further in
either 7H9 broth or on 7H11 solid media for seven to ten days at
37
C for rapidly growing mycobacteria or two to four weeks at
37
C for slowly growing mycobacteria. A preference to increase
the number of inoculated cultures, as opposed to increasing the
growth time, was suggested to limit the amount of degraded DNA
resulting from older cells that could ultimately impact overall
sequence quality and read lengths [28]. Genomic DNA prepared
in this manner is also amenable to next-generation short-read
sequencing on platforms such as Illumina [29] or amplicon-based
Sanger sequencing.
3.3 Nucleic Acid
Quality
For the derived genomic sequence to be as reliable and as informative as possible, the sample of DNA to be sequenced should be of
the highest quality possible. The primary quality metrics that affect
sequencing are the concentration of DNA, its purity from contaminating chemicals and biomolecules, and potentially DNA fragment
length.
3.3.1 Concentration
For WGS to be successful, there must clearly be a sufficient quantity
of DNA in the sample to be sequenced. The two most common
methods for DNA quantification include spectrophotometry and
fluorescence quantitation. DNA spectrophotometry uses the absorbance of ultraviolet wavelengths (260 nm) to measure the concentration of nucleic acid in a sample. However, since DNA (both
single-stranded and double-stranded) and RNA absorb ultraviolet
light at this wavelength, contamination of the desired DNA with
RNA that was not degraded during the extraction will increase the
absorbance and thus the apparent concentration, leading to an
overestimation of the actual quantity of DNA in the sample. This
could lead to adding less DNA to the library prep process than is
optimal, resulting in a poor sequencing reaction.
408
Marco A. Riojas et al.