TITLE: Ultrasensitive hybridization capture of short tuberculosis cell-free DNA from urine
AUTHORS: Amy Oreskovic, Barry Lutz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Overview: This protocol describes a method for highly sensitive sequence-specific purification of short tuberculosis (TB) urine cell-free DNA (cfDNA) from large-volume (10 mL) urine samples. </span><span>Biotinylated oligonucleotide capture probes complementary to the target of interest are immobilized on streptavidin-coated magnetic beads and used to capture, concentrate, and purify target DNA via hybridization. This protocol improves upon the analytical performance of both existing silica-based extraction methods for urine cfDNA and previous hybridization capture protocols, and meets several previously unmet design criteria for urine cfDNA: (1) </span><span style = "font-weight:bold;">high recovery of short fragments</span><span>, (2) </span><span style = "font-weight:bold;">large sample input volume</span><span>, and (3) </span><span style = "font-weight:bold;"><1 copy/mL sensitivity</span><span>. There are two key innovations that contribute to the robustness and unprecedented sensitivity of this method: </span><span style = "font-weight:bold;">dual biotinylated capture probes</span><span>, which increase recovery compared to single biotinylated probes by moderating probe density on the bead surface (a key variable affecting efficiency of surface-based hybridization), improving thermostability of the bead-probe linkage, and eliminating interference by endogenous biotin in urine, and a </span><span style = "font-weight:bold;">two-probe system</span><span> for each target region, which enables recovery of both strands of double-stranded DNA. We designed this hybridization method for capture of short, dilute TB cfDNA fragments from urine, but anticipate that it will be versatile and may be useful for other applications and sample types requiring sensitive and efficient purification of DNA from large sample volumes. Design of primer and probe sequences for new targets is straightforward (see "Materials" tab).</span></div><div class = "text-block"><span style = "font-weight:bold;">Expected performance:</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Near 100% recovery (95% CI: 82.6 - 117.6%) of synthetic ssDNA or dsDNA spiked into 10 mL urine samples, verified across concentrations of at least 1 – 10,000 copies/mL and fragment lengths of at least 25 – 150 bp</li><li style = "counter-reset:ol0;list-style-type:disc;">Limit of detection of ≤5 copies of dsDNA in 10 mL urine (0.5 copies/mL)</li><li style = "counter-reset:ol0;list-style-type:disc;">Enables amplification of DNA from 10 mL urine in a single PCR well (500X concentration factor)</li><li style = "counter-reset:ol0;list-style-type:disc;">Tolerant to variations in sample composition, including pH (5 – 8), salt (0 – 500 mM), and non-target DNA (0 – 10 µg)</li></ul></div><div class = "text-block"><span style = "font-weight:bold;">Graphic abstract: (A)</span><span> Schematic of capture probe design. </span><span style = "font-weight:bold;">(B)</span><span> Overview of workflow.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/c7he86mw.jpg" /></div></div>

[STEPS]
?. [Urine collection and storage]
Before urine collection, prepare 15 mL Eppendorf DNA LoBind tubes with 500 μL 0.5 M EDTA and 100 μL 1M Tris-HCl pH 8.
?. [Urine collection and storage]
Collect urine sample in a sterile container.
?. [Urine collection and storage]
Immediately after urine collection, add 10 mL of urine to each prepared 15 mL tube. The final concentration of EDTA and Tris-HCl will be ~25 mM and ~10 mM, respectively.
CRITICAL STEP: EDTA protects cfDNA from degradation by inactivating DNAses in urine. Tris-HCl buffers urine pH for storage. To minimize the risk of cfDNA degradation, it is important to add EDTA and Tris-HCl as soon as possible after urine collection.
?. [Urine collection and storage]
Mix by inversion.
?. [Urine collection and storage]
Freeze at -80°C until analysis.
?. [Urine collection and storage]
Immediately before analysis, thaw urine at 37°C.
?. [Urine collection and storage]
Mix urine gently by inversion.
?. [Urine collection and storage]
Test with Fisherbrand 10-SG Urine Reagent Strips (optional).
?. [Urine collection and storage]
Centrifuge urine for 5 minutes at 8000g to pellet cell debris.
?. [Urine collection and storage]
Transfer cell-free urine supernatant to new 15 mL Eppendorf DNA LoBind tubes.
Alternative:  If possible at collection site, can centrifuge and transfer cell-free urine to new tubes prior to freezing.
?. [Immobilize capture probes on magentic beads]
Vortex Dynabeads MyOne Streptavidin C1 for 30 seconds to ensure that beads are evenly dispersed in solution.
?. [Immobilize capture probes on magentic beads]
Pipette beads into a 1.5 mL Eppendorf DNA LoBind tube. Prepare 50 μL (0.5 mg) beads per 10 mL urine sample to be analyzed (eg, for 16 samples, prepare 800 μL beads).
?. [Immobilize capture probes on magentic beads]
Wash beads three times with an equal volume of high salt wash buffer (1M NaCl, 10 mM Tris-HCl pH 8, 0.5% Tween-20).
?. [Immobilize capture probes on magentic beads]
Place beads on magnetic rack for 1 minute.
?. [Immobilize capture probes on magentic beads]
Remove and discard supernatant by pipette.
?. [Immobilize capture probes on magentic beads]
Add equal volume of high salt wash buffer (1M NaCl, 10 mM Tris-HCl pH 8, 0.05% Tween-20).
?. [Immobilize capture probes on magentic beads]
Vortex for 5 seconds.
?. [Immobilize capture probes on magentic beads]
Spin down briefly.
?. [Immobilize capture probes on magentic beads]
Repeat twice for a total of three washes.
?. [Immobilize capture probes on magentic beads]
Resuspend in equal volume of high salt wash buffer (1M NaCl, 10 mM Tris-HCl pH 8, 0.05% Tween-20).
?. [Immobilize capture probes on magentic beads]
Pre-mix dual biotinylated capture probes BP1 and BP2 in TLE to a final concentration of 50 μM BP1 and 50 μM BP2.
Note: We recommend pre-mixing probes in bulk ahead of time, aliquoting, and freezing at -20°C until use.
?. [Immobilize capture probes on magentic beads]
Add pre-mixed biotinylated capture probes BP1 and BP2 to side wall of bead tube, close lid, and immediately vortex  for 5 seconds. Use 0.5 μL of pre-mixed 50 μM probes (25 pmol of each probe) per 50 μL beads (eg, use 8 μL probe mix for 16 samples).
CRITICAL STEP: It is important to pre-mix the probes and vortex the beads immediately to ensure homogenous probe distribution on the beads. If probes are added sequentially, binding to the beads and subsequent recovery will favor the probe that is added first.
?. [Immobilize capture probes on magentic beads]
Rotate for 15 minutes at room temperature to immobilize probes on beads.
?. [Immobilize capture probes on magentic beads]
Spin down briefly.
?. [Immobilize capture probes on magentic beads]
Wash beads three times with an equal volume of high salt wash buffer (1M NaCl, 10 mM Tris-HCl pH 8, 0.5% Tween-20).
?. [Immobilize capture probes on magentic beads]
Place beads on magnetic rack for 1 minute.
?. [Immobilize capture probes on magentic beads]
Remove and discard supernatant by pipette.
?. [Immobilize capture probes on magentic beads]
Add equal volume of high salt wash buffer (1M NaCl, 10 mM Tris-HCl pH 8, 0.05% Tween-20).
?. [Immobilize capture probes on magentic beads]
Vortex for 5 seconds.
?. [Immobilize capture probes on magentic beads]
Spin down briefly.
?. [Immobilize capture probes on magentic beads]
Repeat twice for a total of three washes.
?. [Immobilize capture probes on magentic beads]
Resuspend beads in an equal volume high salt wash buffer (1M NaCl, 10 mM Tris-HCl pH 8, 0.05% Tween-20).
Alternative: Beads can be prepared ahead of time and stored at 4°C for 1 - 2 weeks. If storing for future use, resuspend beads in an equal volume 1X TLE with 0.5% Tween-20 instead of high salt wash buffer and store at 4°C.
?. [Capture TB urine cfDNA by hybridization]
Add 2.5 mL 5 M NaCl (final concentration 1 M), 127 μL 10% Tween-20 (final concentration 0.1%) and 50 μL prepared beads to each 10 mL sample. Vortex beads well to ensure that they are evenly resuspended before adding to urine.
?. [Capture TB urine cfDNA by hybridization]
If spiking in positive control, add it now.
?. [Capture TB urine cfDNA by hybridization]
Mix well by inversion.
?. [Capture TB urine cfDNA by hybridization]
Denature for 15 minutes in dry bath with 15 mL tube block preheated to 120°C (urine temperature should reach >90°C).
Alternative: Denature in 95°C water bath for 15 minutes. CRITICAL STEP: Reaching a sufficient denaturing temperature is critical for maximum recovery.
?. [Capture TB urine cfDNA by hybridization]
Rotate for 30 minutes at room temperature to hybridize TB-specific cfDNA to capture probes.
?. [Wash to remove urine inhibitors and non-target DNA]
Centrifuge for 5 minutes at 5000g to pellet beads.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove and discard all but approximately 1 mL supernatant using 10 mL serological pipette.
?. [Wash to remove urine inhibitors and non-target DNA]
Resuspend beads in remaining supernatant using P1000 pipette and transfer to 1.5 mL Eppendorf DNA LoBind tube.
?. [Wash to remove urine inhibitors and non-target DNA]
Place on magnetic rack for 1 minute.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove and discard supernatant by pipette.
Note: Some urine samples (often those with blood or abnormally high protein concentrations) may cause beads to aggregate poorly on the magnet. If this is the case, we suggest leaving the sample on the magnet for longer (5 min) and/or using a minicentrifuge to help pellet the beads before removing the supernatant here and in subsequent wash steps. It is okay to leave more residual volume to avoid bead loss. This problem occurs infrequently, but we have still successfully extracted cfDNA from samples despite increased residual volumes and bead loss during the wash steps.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove tube from magnetic rack.
?. [Wash to remove urine inhibitors and non-target DNA]
Add 1 mL high salt wash buffer (1 M NaCl, 10 mM Tris-HCl pH 8, 0.05% Tween-20) and wash by inverting 10-20 times, or until no bead aggregate is left on tube wall. Do not vortex.
?. [Wash to remove urine inhibitors and non-target DNA]
Spin down briefly.
?. [Wash to remove urine inhibitors and non-target DNA]
Place on magnetic rack for 1 minute.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove and discard supernatant by pipette.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove tube from magnetic rack.
?. [Wash to remove urine inhibitors and non-target DNA]
Add 1 mL high salt wash buffer (1 M NaCl, 10 mM Tris-HCl pH 8, 0.05% Tween-20) and wash by inverting 10-20 times, or until no bead aggregate is left on tube wall. Do not vortex.
?. [Wash to remove urine inhibitors and non-target DNA]
Spin down briefly.
?. [Wash to remove urine inhibitors and non-target DNA]
Place on magnetic rack for 1 minute.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove and discard supernatant by pipette.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove tube from magnetic rack.
?. [Wash to remove urine inhibitors and non-target DNA]
Add 1 mL low salt wash buffer (15 mM NaCl, 10 mM Tris-HCl pH 8, no Tween-20) and wash by inverting 10-20 times, or until no bead aggregate is left on tube wall. Do not vortex.
?. [Wash to remove urine inhibitors and non-target DNA]
Spin down briefly.
?. [Wash to remove urine inhibitors and non-target DNA]
Place on magnetic rack for 1 minute.
?. [Wash to remove urine inhibitors and non-target DNA]
Remove and discard supernatant by pipette.
?. [Wash to remove urine inhibitors and non-target DNA]
Spin down again.
?. [Wash to remove urine inhibitors and non-target DNA]
Place on magnetic rack and remove as much liquid as possible using P20 pipette.
CRITICAL STEP: Because of the small elution volume in the next step, it is important to remove as much of the residual wash buffer as possible, while avoiding bead loss.
?. [Elute purified TB cfDNA]
Prepare fresh 20 mM NaOH by adding 400 μL water to 100 μL 100 mM NaOH.
?. [Elute purified TB cfDNA]
Add 20 μL freshly-prepared 20 mM NaOH to beads to elute purified TB-specific cfDNA.
?. [Elute purified TB cfDNA]
Vortex for 5 seconds.
?. [Elute purified TB cfDNA]
Spin down briefly.
?. [Elute purified TB cfDNA]
Place on magnetic rack.
?. [Elute purified TB cfDNA]
Transfer as much supernatant as possible (usually 20 – 21 μL; set P20 pipette to maximum volume) directly to PCR well or to new Eppendorf DNA LoBind tube. This contains purified TB-specific cfDNA.
CRITICAL STEP: To maximize recovery, make sure to transfer as much eluate as possible to PCR. Avoid transferring any beads to PCR because they can lead to PCR inhibition. Note: Some urine samples (often those with blood or abnormally high protein concentrations) may also make it difficult to avoid bead transfer to PCR. If this is the case, we suggest leaving the sample on the magnet for longer (5 min) and/or using a minicentrifuge to help pellet the beads before eluting. It is okay if the eluted volume is This problem occurs infrequently, but we have still successfully detected cfDNA in samples despite reduced elution volumes and PCR inhibition caused by minor transfer of beads to PCR.
?. [Elute purified TB cfDNA]
Partially neutralize with 3.5 μL 100 mM HCl.
Note: Since the exact elution volume varies across samples, the PCR buffer will adjust to final pH, and tolerates sligtly basic pH better than acidic pH. For this reason, we also recommend using PCR conditions with a high buffering capacity (eg, 80 mM Tris-SO4 used here).
?. [Elute purified TB cfDNA]
Proceed directly to PCR.
Alternative: We recommend proceeding directly to PCR, but if you cannot amplify immediately, add 2 μL 10X TLE and store at 4°C.
?. [Quantify by PCR]
Prepare a PCR master mix according to the table below. Each reaction should contain 1.25 U OneTaq Hot Start DNA Polymerase (NEB), 1X NEB OneTaq GC Reaction Buffer (80 mM Tris-SO4, 20 mM (NH4)2SO4, 2 mM MgSO4, 5% glycerol, 5% DMSO, 0.06% IGEPAL CA-630, 0.05% Tween-20, pH 9.2), 0.8 mM dNTPs (NEB), 0.4X EvaGreen (Biotium), 200 nM forward primer, and 200 nM reverse primer. The total reaction volume should be 50 μL, 26 μL of which is master mix.
?. [Quantify by PCR]
For the hybridization output, add 26 μL master mix to the entire neutralized output (~24 μL) of each experimental sample.
?. [Quantify by PCR]
For the standard curve (0, 10, 102, 103, 104, and 105 copies positive control, in triplicate), add 19 μL nuclease-free water and 26 μL master mix to 5 μL DNA standard.
?. [Quantify by PCR]
Amplify in CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with an initial incubation of 94°C for 3 min followed by 45 amplification cycles (94°C for 30s, 64°C for 30s, and 68°C for 1 min).
?. [Quantify by PCR]
Measure amplicon Tm by post-amplification melt curve analysis from 65°C to 95°C in 0.5°C increments every 5 seconds.
?. [Quantify by PCR]
Determine Cq values at a threshold of 500 RFU and calculate recovered copies using the standard curve.
Recovery of the positive control should be near 100% (80 - 120%). Calculated percent recovery may occasionally be >100% due to inherent variability of PCR; a small shift in Cq (positive control or PCR standards) leads to an exponential change in calculated percent recovery. The positive control PCR amplification curve should be similar to the corresponding PCR standard.There should be no amplification of the negative control or PCR NTCs up to at least 45 cycles. Detected copy number in experimental samples will depend on the target of interest and may vary by several orders of magnitude across biological replicates. In preliminary clinical testing with our IS6110 TB target, we observed copy numbers ranging from  in 10 mL urine (median 147 copies).
?. [Quantify by PCR]
Confirm that amplicon Tm for clinical samples matches that of expected target (should be 0.5°C below the positive control Tm).
The Tm of TB-positive samples should be ~0.5°C below the Tm of the positive control. If any samples amplify with incorrect Tm, they should be considered TB-negative.