(e.g., Qubit) as well as to determine the average fragment size of
each library using quantitative platforms such as the Agilent TapeStation 4200 or the Agilent Fragment Analyzer. This information is
also used for normalization of each library that will be loaded on
the Illumina instrument. In the final sample preparation step, the
samples are pooled together, treated with sodium hydroxide to
denature the libraries into single-stranded DNA, and loaded for
sequencing.
3.4.2 Oxford Nanopore
Oxford Nanopore Technologies (ONT) genomic DNA library
preparation is carried out using one or more of the several available
kits, depending on the specific application. Because protocols covering the variety of different kits offered by ONT cannot be covered
in a single summary section, this section will instead describe generally the protocols employed during library prep using the Ligation Sequencing Kit (SQK-LSK109) and the Native Barcoding
Expansion (EXP-NBD104).
As with Illumina sequencing, the DNA is first quantified before
the start of the library preparation. As described in previous sections, minimizing shearing is essential in order to properly generate
long sequencing reads. Thus, it is highly advisable to mix reaction
solutions at all steps which contain DNA via gentle flicking or
tapping of the sample tube rather than vortexing. The current
library preparation consists of three separate steps: DNA repair/
end-prep, native barcode addition, and adapter ligation.
The DNA is bound to magnetic beads after each step and
washed with 80% ethanol to remove buffer salts, enzymes, or
other substrates, which could affect downstream usage. Altering
the bead:sample ratio used allows for size selection of either larger
or smaller fragments. A lower relative volume of magnetic beads
leads to greater recovery of higher fragment sizes, while a higher
relative volume of beads leads to greater recovery of smaller fragment sizes. A 1Â magnetic bead ratio is standard for recovering all
fragments within a sample. Ethanol is used to wash the samples due
to its ability to solubilize buffer salts while minimizing the solubility
of the DNA. Because ethanol concentrations below 70% allow for
elution of the DNA, 80% ethanol is used to prevent this possibility.
It is therefore crucial that all ethanol is removed from the magnetic
beads before the addition of an elution buffer. This is ensured by a
30-s open-air drying of the magnetic beads to allow for ethanol
evaporation. DNA is then simultaneously end-prepped and
repaired using an FFPE DNA repair mix (designed for use with
formalin-fixed paraffin-embedded samples) and end repair/dAtailing module reagents. This ensures fragments possess blunt
ends containing both 5
0 phosphate and 3
0 hydroxyl groups, which
are necessary for subsequent adapter ligation. The DNA is then
bound to the magnetic beads at a 1Â ratio and washed with 80%
ethanol.
Mycobacterial ID and Characterization Using WGS
411
each library using quantitative platforms such as the Agilent TapeStation 4200 or the Agilent Fragment Analyzer. This information is
also used for normalization of each library that will be loaded on
the Illumina instrument. In the final sample preparation step, the
samples are pooled together, treated with sodium hydroxide to
denature the libraries into single-stranded DNA, and loaded for
sequencing.
3.4.2 Oxford Nanopore
Oxford Nanopore Technologies (ONT) genomic DNA library
preparation is carried out using one or more of the several available
kits, depending on the specific application. Because protocols covering the variety of different kits offered by ONT cannot be covered
in a single summary section, this section will instead describe generally the protocols employed during library prep using the Ligation Sequencing Kit (SQK-LSK109) and the Native Barcoding
Expansion (EXP-NBD104).
As with Illumina sequencing, the DNA is first quantified before
the start of the library preparation. As described in previous sections, minimizing shearing is essential in order to properly generate
long sequencing reads. Thus, it is highly advisable to mix reaction
solutions at all steps which contain DNA via gentle flicking or
tapping of the sample tube rather than vortexing. The current
library preparation consists of three separate steps: DNA repair/
end-prep, native barcode addition, and adapter ligation.
The DNA is bound to magnetic beads after each step and
washed with 80% ethanol to remove buffer salts, enzymes, or
other substrates, which could affect downstream usage. Altering
the bead:sample ratio used allows for size selection of either larger
or smaller fragments. A lower relative volume of magnetic beads
leads to greater recovery of higher fragment sizes, while a higher
relative volume of beads leads to greater recovery of smaller fragment sizes. A 1Â magnetic bead ratio is standard for recovering all
fragments within a sample. Ethanol is used to wash the samples due
to its ability to solubilize buffer salts while minimizing the solubility
of the DNA. Because ethanol concentrations below 70% allow for
elution of the DNA, 80% ethanol is used to prevent this possibility.
It is therefore crucial that all ethanol is removed from the magnetic
beads before the addition of an elution buffer. This is ensured by a
30-s open-air drying of the magnetic beads to allow for ethanol
evaporation. DNA is then simultaneously end-prepped and
repaired using an FFPE DNA repair mix (designed for use with
formalin-fixed paraffin-embedded samples) and end repair/dAtailing module reagents. This ensures fragments possess blunt
ends containing both 5
0 phosphate and 3
0 hydroxyl groups, which
are necessary for subsequent adapter ligation. The DNA is then
bound to the magnetic beads at a 1Â ratio and washed with 80%
ethanol.
Mycobacterial ID and Characterization Using WGS
411