2.2 DNA Shearing
1. Covaris E210 Focused-ultrasonicator.
2. Covaris microTUBE with AFA fiber and snap cap (520045,
Covaris).
3. TE LOW buffer (10 mM Tris–HCl, pH. 8.0, 0.1 mM EDTA).
4. Human genomic DNA (see Note 1).
5. DNA LoBind 1.5 mL tubes (Eppendorf).
6. Microfuge.
2.3 Postshearing
Sample Purification
1. Agencourt AMPure XP beads (Beckman Coulter).
2. DynaMag-2 magnetic stand (Life Technologies).
3. 70% ethanol (see Note 2).
4. Nuclease-free water.
5. DNA LoBind 1.5 mL tubes (Eppendorf).
6. Microfuge.
7. Heat block set at 37 ˚C.
8. 2200 TapeStation Nucleic Acid System (Agilent).
9. D1000 ScreenTape (Agilent) and D1000 reagents (Agilent).
10. Thin-wall 8-strip tubes and caps (Thermo Scientific).
2.4 End Repair,
A-Tailing, and Adapter
Ligation
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent) (see Note 3).
2. End Repair mix (52μL per sample): 35.2μL of Nuclease-Free
Water, 10μL of 10Â End Repair Buffer, 1.6μL of dNTP mix,
1μL of T4 DNA polymerase, 2μL of Klenow DNA polymerase,
and 2.2μL of T4 Polynucleotide kinase.
3. A-Tailing Mix (20μL per sample): 11μL of Nuclease-Free
Water, 12μL of 10Â Klenow DNA polymerase buffer, 1μL of
ATP, and 3μL of Exo (À) klenow DNA polymerase.
4. Adapter Ligation Mix (37μL per sample): 15.5μL of NucleaseFree Water, 10μL of 5Â T4 DNA ligase buffer, 10μL of Adaptor Oligo mix, and 1.5μL of T4 DNA ligase.
5. Agencourt AMPure XP beads (Beckman Coulter).
6. DynaMag-96 Side magnet (Life Technologies).
7. 70% ethanol (see Note 2).
8. Nuclease-free water.
9. Thin-wall 8-strip tubes and caps (Thermo Scientific).
10. 2200 TapeStation Nucleic Acid System (Agilent).
11. D1000 ScreenTape and D1000 reagents (Agilent).
12. Thermal cycler, e.g., SureCycler 8800 with 96-well module.
13. Microfuge.
14. 5-50μL 8-channel pipette and 20–200μL 8-channel pipette.
Direct WGS of M. tb’
463
1. Covaris E210 Focused-ultrasonicator.
2. Covaris microTUBE with AFA fiber and snap cap (520045,
Covaris).
3. TE LOW buffer (10 mM Tris–HCl, pH. 8.0, 0.1 mM EDTA).
4. Human genomic DNA (see Note 1).
5. DNA LoBind 1.5 mL tubes (Eppendorf).
6. Microfuge.
2.3 Postshearing
Sample Purification
1. Agencourt AMPure XP beads (Beckman Coulter).
2. DynaMag-2 magnetic stand (Life Technologies).
3. 70% ethanol (see Note 2).
4. Nuclease-free water.
5. DNA LoBind 1.5 mL tubes (Eppendorf).
6. Microfuge.
7. Heat block set at 37 ˚C.
8. 2200 TapeStation Nucleic Acid System (Agilent).
9. D1000 ScreenTape (Agilent) and D1000 reagents (Agilent).
10. Thin-wall 8-strip tubes and caps (Thermo Scientific).
2.4 End Repair,
A-Tailing, and Adapter
Ligation
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent) (see Note 3).
2. End Repair mix (52μL per sample): 35.2μL of Nuclease-Free
Water, 10μL of 10Â End Repair Buffer, 1.6μL of dNTP mix,
1μL of T4 DNA polymerase, 2μL of Klenow DNA polymerase,
and 2.2μL of T4 Polynucleotide kinase.
3. A-Tailing Mix (20μL per sample): 11μL of Nuclease-Free
Water, 12μL of 10Â Klenow DNA polymerase buffer, 1μL of
ATP, and 3μL of Exo (À) klenow DNA polymerase.
4. Adapter Ligation Mix (37μL per sample): 15.5μL of NucleaseFree Water, 10μL of 5Â T4 DNA ligase buffer, 10μL of Adaptor Oligo mix, and 1.5μL of T4 DNA ligase.
5. Agencourt AMPure XP beads (Beckman Coulter).
6. DynaMag-96 Side magnet (Life Technologies).
7. 70% ethanol (see Note 2).
8. Nuclease-free water.
9. Thin-wall 8-strip tubes and caps (Thermo Scientific).
10. 2200 TapeStation Nucleic Acid System (Agilent).
11. D1000 ScreenTape and D1000 reagents (Agilent).
12. Thermal cycler, e.g., SureCycler 8800 with 96-well module.
13. Microfuge.
14. 5-50μL 8-channel pipette and 20–200μL 8-channel pipette.
Direct WGS of M. tb’
463