For quantitative QC of DNA strand length, various commercial
platforms can provide an indication of the distribution of fragment
sizes. Such platforms include the Agilent TapeStation and the
Agilent Fragment Analyzer. These systems provide output as a
fragment size distribution graph showing the relative quantity of
DNA per fragment length, as well as the calculated mean fragment
size and peak fragment size.
3.4 Library
Preparation
3.4.1 Illumina
For sequencing on Illumina platforms, samples are first measured
for concentration using a fluorescence-based system, e.g., Qubit
Broad Range Assay (Life Technologies). Based on the measured
concentration, between 1 and 500 ng of total genomic DNA is
used as input for the library prep kit. While there are dozens of
library prep kits on the market, the authors prefer the Nextera DNA
Flex Library Prep Kit (Illumina) due to its ability to achieve relatively even sequencing coverage across a target, even with low or
high GC content templates such as mycobacteria. The samples are
first “Tagmented” enzymatically by incubation with magnetic
bead-linked transposomes. This results in the genomic DNA
being simultaneously fragmented at random locations and tagged
with universal adapter sequences, which allows for incorporation of
unique indices during the subsequent short-cycle PCR. The randomly fragmented DNA remains bound to the magnetic beads,
and, after treatment with a buffer that neutralizes the tagmentation
reaction, the DNA-bead complexes are washed before PCR enrichment. Using a standardized tagmentation incubation time leads to
creation of libraries with an appropriate range of fragment sizes,
which is necessary for the subsequent sequencing-by-synthesis that
occurs on the flow cell.
The following short-cycle PCR serves to enrich the quantity of
the genomic DNA library that was generated in the previous step
and allows for incorporation of unique indices at the distal ends of
the DNA fragments, which will be used during postsequencing
analysis to identify multiple samples that are multiplexed on the
flow cell. The number of PCR cycles used is determined by the
amount of starting DNA input. For the higher end of the range
(50–500 total ng genomic DNA), as few as 5 cycles are sufficient for
enrichment, while 12 cycles are recommended for the lowest end of
the range (1–9 total ng).
After PCR, the enriched libraries are separated from the original beads from the tagmentation step, and the libraries are further
size-selected with the addition of specific volumes of DNA-binding
magnetic beads. Two separate size-selection steps are performed,
removing undesired very large and very small fragments, respectively. The samples are given a final wash with 80% ethanol to
eliminate all enzymes, buffers, and unincorporated dNTPs from
previous steps and are then eluted from the magnetic beads. The
purified libraries undergo quality control checks for concentration
410
Marco A. Riojas et al.
platforms can provide an indication of the distribution of fragment
sizes. Such platforms include the Agilent TapeStation and the
Agilent Fragment Analyzer. These systems provide output as a
fragment size distribution graph showing the relative quantity of
DNA per fragment length, as well as the calculated mean fragment
size and peak fragment size.
3.4 Library
Preparation
3.4.1 Illumina
For sequencing on Illumina platforms, samples are first measured
for concentration using a fluorescence-based system, e.g., Qubit
Broad Range Assay (Life Technologies). Based on the measured
concentration, between 1 and 500 ng of total genomic DNA is
used as input for the library prep kit. While there are dozens of
library prep kits on the market, the authors prefer the Nextera DNA
Flex Library Prep Kit (Illumina) due to its ability to achieve relatively even sequencing coverage across a target, even with low or
high GC content templates such as mycobacteria. The samples are
first “Tagmented” enzymatically by incubation with magnetic
bead-linked transposomes. This results in the genomic DNA
being simultaneously fragmented at random locations and tagged
with universal adapter sequences, which allows for incorporation of
unique indices during the subsequent short-cycle PCR. The randomly fragmented DNA remains bound to the magnetic beads,
and, after treatment with a buffer that neutralizes the tagmentation
reaction, the DNA-bead complexes are washed before PCR enrichment. Using a standardized tagmentation incubation time leads to
creation of libraries with an appropriate range of fragment sizes,
which is necessary for the subsequent sequencing-by-synthesis that
occurs on the flow cell.
The following short-cycle PCR serves to enrich the quantity of
the genomic DNA library that was generated in the previous step
and allows for incorporation of unique indices at the distal ends of
the DNA fragments, which will be used during postsequencing
analysis to identify multiple samples that are multiplexed on the
flow cell. The number of PCR cycles used is determined by the
amount of starting DNA input. For the higher end of the range
(50–500 total ng genomic DNA), as few as 5 cycles are sufficient for
enrichment, while 12 cycles are recommended for the lowest end of
the range (1–9 total ng).
After PCR, the enriched libraries are separated from the original beads from the tagmentation step, and the libraries are further
size-selected with the addition of specific volumes of DNA-binding
magnetic beads. Two separate size-selection steps are performed,
removing undesired very large and very small fragments, respectively. The samples are given a final wash with 80% ethanol to
eliminate all enzymes, buffers, and unincorporated dNTPs from
previous steps and are then eluted from the magnetic beads. The
purified libraries undergo quality control checks for concentration
410
Marco A. Riojas et al.