4 Notes
1. This protocol describes the use of the Agilent SureSelect
XT 3μg
input methodology; in the majority of cases, less than 3μg of
M. tuberculosis DNA is obtained following extraction from
clinical samples, and we therefore use human DNA as a carrier
to bulk the sample to 3μg; however, users may prefer to use the
200 ng method, which uses a smaller shearing volume (50μL)
and dilution of adapters 1:10; see http://www.chem.agilent.
com/library/usermanuals/Public/G7530-90000.pdf for full
details.
2. 70% ethanol should be made fresh daily using 100% molecular
grade ethanol and ultrapure, molecular biology grade water.
3. This protocol describes the use of the Agilent SureSelect
XT 3μg
methodology; however, since we first established this protocol,
other kits have come to market, including the SureSeq NGS
Library Preparation Kit (500,070, Oxford Gene Technology),
which requires less bead cleanup steps.
4. 120-mer RNA baits spanning the length of the positive strand
of GenBank M. tuberculosis H37Rv reference genomes were
designed using an in-house PERL script developed by the
PATHSEEK consortium. The specificity of the baits was verified by BLASTn searches against the Human Genomic Transcript database. The custom-designed M. tuberculosis bait
library was uploaded to SureDesign and synthesized by Agilent
Technologies. Baits can be purchased from OGT.
5. 10 N NaOH should be diluted to 0.2 N fresh, just before use.
6. There is potential, at this stage, to run samples in parallel—with
and without culturing—if such a comparison is sought. If so,
0.1 mL of the decontaminated sputum can now be taken for
inoculation into culture medium, as per user requirements. If
this is followed, the remaining 0.2 mL should be diluted with a
further 0.1 mL PBS prior to DNA extraction, in order to
supply the required volume for DNA extraction (0.3 mL).
Whole-genome sequencing of DNA extracted from cultured
M. tuberculosis can be prepared as per the method given here
for sputum samples, but the enrichment hybridization step
(Sub-heading 3.6) is not required, as the DNA extracted
from a culture-grown sample will be pure M. tuberculosis, so
can go straight from Sub-heading 3.5straight to 3.7.
7. Flow-through contains Buffer AL or Buffer AW1 and is not
compatible with bleach.
8. Add fresh deionized water to the Covaris tank, turn on chiller,
and allow it to reach 5
C before use.
Direct WGS of M. tb’
477
1. This protocol describes the use of the Agilent SureSelect
XT 3μg
input methodology; in the majority of cases, less than 3μg of
M. tuberculosis DNA is obtained following extraction from
clinical samples, and we therefore use human DNA as a carrier
to bulk the sample to 3μg; however, users may prefer to use the
200 ng method, which uses a smaller shearing volume (50μL)
and dilution of adapters 1:10; see http://www.chem.agilent.
com/library/usermanuals/Public/G7530-90000.pdf for full
details.
2. 70% ethanol should be made fresh daily using 100% molecular
grade ethanol and ultrapure, molecular biology grade water.
3. This protocol describes the use of the Agilent SureSelect
XT 3μg
methodology; however, since we first established this protocol,
other kits have come to market, including the SureSeq NGS
Library Preparation Kit (500,070, Oxford Gene Technology),
which requires less bead cleanup steps.
4. 120-mer RNA baits spanning the length of the positive strand
of GenBank M. tuberculosis H37Rv reference genomes were
designed using an in-house PERL script developed by the
PATHSEEK consortium. The specificity of the baits was verified by BLASTn searches against the Human Genomic Transcript database. The custom-designed M. tuberculosis bait
library was uploaded to SureDesign and synthesized by Agilent
Technologies. Baits can be purchased from OGT.
5. 10 N NaOH should be diluted to 0.2 N fresh, just before use.
6. There is potential, at this stage, to run samples in parallel—with
and without culturing—if such a comparison is sought. If so,
0.1 mL of the decontaminated sputum can now be taken for
inoculation into culture medium, as per user requirements. If
this is followed, the remaining 0.2 mL should be diluted with a
further 0.1 mL PBS prior to DNA extraction, in order to
supply the required volume for DNA extraction (0.3 mL).
Whole-genome sequencing of DNA extracted from cultured
M. tuberculosis can be prepared as per the method given here
for sputum samples, but the enrichment hybridization step
(Sub-heading 3.6) is not required, as the DNA extracted
from a culture-grown sample will be pure M. tuberculosis, so
can go straight from Sub-heading 3.5straight to 3.7.
7. Flow-through contains Buffer AL or Buffer AW1 and is not
compatible with bleach.
8. Add fresh deionized water to the Covaris tank, turn on chiller,
and allow it to reach 5
C before use.
Direct WGS of M. tb’
477