The authors thank Y. Zhu for her comments on the manuscript. Research reported in this publication was partially supported by the National Cancer Institute of the National Institutes of Health under Award Number P30CA034196. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTE: The NLR-seq protocol consists of three consecutive steps: 1) extraction of high-molecular weight (HMW) genomic DNA; 2) ultra-long DNA library construction, which includes fragmentation of the HMW DNA into the desired sizes and ligation of sequencing adapters to the DNA ends; and 3) loading of the adapter-ligated DNA onto the arrays of nanopores (Figure 1).
1. HMW DNA extraction
<ol>
	<li>Reagent setup. Make 1x phosphate buffered saline (PBS) buffer (1,000 mL) by adding 100 mL of PBS (10x) to 900 mL of water and mix well. Make lysis buffer (50&#160;mL) by adding 43.5 mL of water to a 50 mL tube. Add 500 &#956;L of Tris (1 M, pH 8.0), 1 mL of sodium chloride (NaCl) (5 M), 2.5 mL of ethylenediaminetetraacetic acid (EDTA) (0.5 M, pH 8.0) and 2.5 mL of sodium dodecyl sulfate (SDS) (10%, wt/vol) to the tube and mix well. 
	NOTE: This PBS buffer can be stored at 4 &#176;C for up to 6 months. The premade lysis buffer can be stored at RT for up to 2 months.</li>
	<li>Check the cell mortality and count the cells. Ensure that the live ratio is &#62; 85% and the total cell number is 30 x 106. 
	NOTE: The cells used in this protocol are from the HG00733 cell line, a human lymphoblastoid cell line of Puerto Rican origin widely used in the 1000 Genome consortium for structural variation analysis (see table of materials for ordering information), which belongs to International Genome Sample Resource.</li>
	<li>Collect the cells by centrifuging at 200 x g for 5 min at RT. Discard the medium and resuspend the cell pellet (30 x 106 cells) with 5 mL of 1x PBS buffer. Centrifuge again at 200 x g for 5 min at RT and discard the supernatant. 
	NOTE: 25-35 x 106 of cells are acceptable for this approach. Further variation in the amount of cells used will need further optimization. The cell pellet can be stored at &#8722;80 &#176;C for up to 6 months.</li>
	<li>Resuspend the cell pellet in 200 &#956;L of 1x PBS buffer. If using a frozen cell pellet, wash with 5 mL of 1x PBS buffer. Centrifuge the solution at 200 x g for 5 min at RT, discard the supernatant and resuspend the cells in 200 &#956;L of 1x PBS buffer.</li>
	<li>Prepare 10 mL of lysis buffer in a 50 mL tube. Add the 200 &#956;L cell suspension to the lysis buffer and vortex at the highest speed for 3 s. Incubate the solution at 37 &#176;C for 1 h.</li>
	<li>Add 2 &#956;L of RNase A (100 mg/mL) to the lysate. Gently rotate the 50 mL tube to mix the sample. Incubate the solution at 37 &#176;C for 1 h.</li>
	<li>Add 50 &#956;L proteinase K (20 mg/mL) to the lysate. Gently rotate the 50 mL tube to mix the sample. Incubate the solution at 50 &#176;C for 2 h. During incubation, gently mix the sample every 30 min.</li>
	<li>Remove the 50 mL tube from 50 &#176;C and let stand at RT for 5 min.</li>
	<li>Add 10 mL of the phenol layer of phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol) to the lysate and rotate the tube on a rotator mixer (see Table of Materials) at RT in a fume hood at 20 rpm for 10 min. Wrap the tube cap with parafilm to prevent leakage during rotation.</li>
	<li>Prepare two 50 mL gel tubes (see Table of Materials) by centrifuging at 1,500 x g for 2 min at RT. 
	NOTE: The gel forms a stable barrier between the nucleic acid-containing aqueous phase and the organic solvent.</li>
	<li>Pour the sample/phenol solution into one of the prepared 50 mL gel tubes from Step 1.10. Centrifuge the solution at 3,000 x g for 10 min at RT.</li>
	<li>Pour the supernatant into a new 50 mL tube. Add 10 mL of the phenol layer of phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol) and rotate the tube on a rotator mixer at RT in a fume hood at 20 rpm for 10 min.</li>
	<li>Repeat step 1.11 once with the second prepared gel tube.</li>
	<li>Pour the supernatant into a new 50 mL tube. Add 25 mL of ice-cold 100% ethanol and gently rotate the tube by hand until the DNA precipitates (Figure 2). 
	NOTE: The precipitation approach helps to stabilize the HMW DNA.</li>
	<li>Bend a 20 &#956;L tip to make a hook. Carefully take out the HMW DNA with the hook and let the liquid drop off.</li>
	<li>Place the HMW DNA into a 50 mL tube containing 40 mL of 70% ethanol. Wash the DNA by gently inverting the tube 3 times.</li>
	<li>Repeat step 1.15 once to collect DNA from the 70% ethanol tube.</li>
	<li>Place the HMW DNA into a 2 mL tube containing 1.8 mL of 70% ethanol.</li>
	<li>Centrifuge the washed HMW DNA at 10,000 x g for 3 s at RT. Remove as much of the residual ethanol as possible by pipetting. 
	NOTE: Do not disturb the DNA pellet when pipetting the residual ethanol.</li>
	<li>Incubate the 2 mL tube at 37 &#176;C for 10 min with the lid open to dry the sample.
	<ol>
		<li>If continuing with step 2.1 (with mechanical shearing and 1D Ligation Sequencing Kit), add 1 mL of TE (10 mM Tris and 1 mM EDTA, pH 8.0) to the 2 mL tube.</li>
		<li>If continuing with Step 2.2 (with transposase-based fragmentation and Rapid Sequencing Kit), add 200 &#956;L of 10 mM Tris (pH 8.0) with 0.02% Triton X-100. 
		NOTE: Do not disturb the DNA pellet. Letting the tube stand at 4 &#176;C in dark for 48 h will help the sample fully resuspend. The HMW DNA can be stored at 4 &#176;C for up to 2 weeks. Longer storage time or other storage conditions may introduce more short fragments.</li>
	</ol>
	</li>
</ol>
2. Ultra-long DNA library construction
NOTE: There are two ways to construct the ultra-long DNA libraries based on two different shearing methods coupled with nanopore sequencing kits. A mechanical shearing-based library produces data with an N50 of 50-70 kb, taking about 8 h for the library construction. A transposase fragmentation-based library produces an N50 of 90-100 kb data, taking only 90 min for the library construction. The mechanical shearing protocol gives higher yield from the same DNA input using identical versions of sequencing adapter and quality of nanopore flow cells.
<ol>
	<li>Mechanical shearing-based library construction
	<ol>
		<li>Thaw and mix the reagents from the ligation kit (see Table of Materials). Thaw FFPE DNA repair buffer and end repair/dA-tailing buffer on ice, then vortex and spin down to mix. Thaw adapter mix (AMX) and adapter bead binding buffer (ABB) on ice, then pipette and spin down to mix. Thaw running buffer with fuel mix (RBF) and elution buffer (ELB) at RT, then vortex and spin down to mix. Thaw library loading beads (LLB) at RT and pipette to mix before use.
		<ol>
			<li>Once thawed, keep all kit components on ice. Take out the enzymes only when needed. Bring the magnetic beads to RT for use. 
			NOTE: For recommendations on the magnetic beads to use see the table of materials.</li>
		</ol>
		</li>
		<li>Check the quality and quantity of the HMW DNA from step 1.21.1. Pipette out 20 &#956;L of DNA into new 1.5 mL tubes from three different locations in the HMW DNA tube using P200 wide bore tips. Take 1 &#956;L from the three aliquots to detect the concentration using a fluorometer and the quality using a UV reading. Check multiple times to confirm the results. 
		NOTE: The expected results are shown in Figure 3A. The OD260/280 value is approximately 1.9 and the OD260/230 value is approximately 2.3.</li>
		<li>Transfer the remaining 940 &#956;L of HMW DNA into a 50 mL tube cap with a P1000 wide bore tip.</li>
		<li>Aspirate all DNA into a 1 mL syringe without the needle.</li>
		<li>Put the 27 G needle onto the syringe and eject all DNA into the cap gently and slowly (~10 s). Take off the 27 G needle from the syringe.</li>
		<li>Repeat steps 2.1.4 and 2.1.5 for 29 times for a total of 30 passes through the needle. 
		NOTE: The sheared HMW DNA can be stored at 4 &#176;C in the dark for up to 24 h. Quality control (QC) is highly recommended by pulsed-field gel electrophoresis, but it is costly and time consuming. If performing QC on an automated pulse field gel electrophoresis machine use a 5-150 kb protocol for a 20 h run. The expected results are shown in Figure 4.</li>
		<li>Prepare the DNA repair reaction in a 0.2 mL tube by adding 100 &#956;L of sheared HMW DNA (20 &#956;g), 15 &#956;L of FFPE DNA repair buffer, 12 &#956;L of FFPE DNA repair mix, and 16 &#956;L of nuclease-free water. Mix the reaction by flicking gently 6 times and spin down to remove bubbles.</li>
		<li>Incubate the reaction at 20 &#176;C for 60 min. Transfer the sample into a new 1.5 mL tube with a P200 wide bore tip.</li>
		<li>Resuspend the magnetic beads by pipetting or vortexing. Add 143 &#956;L beads (1x) to the DNA repair reaction and mix gently by flicking the tube 6 times. Rotate the tube on a rotator mixer at RT at 20 rpm for 30 min.</li>
		<li>Spin down the sample at 1,000 x g for 2 s at RT. Place the tube on a magnetic rack for 10 min. Keep the tube on the magnetic rack and discard the supernatant.</li>
		<li>Keeping the tube on the magnetic rack, add 400 &#956;L of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol after 30 s.</li>
		<li>Repeat step 2.1.11 once.</li>
		<li>Spin down the sample at 1,000 x g for 2 s at RT. Place the tube back on the magnetic rack. Remove any residual ethanol and air dry for 30 s. Do not over dry the pellet.</li>
		<li>Remove the tube from the magnetic rack and add 103 &#956;L of TE (10 mM Tris and 1 mM EDTA, pH 8.0). Gently flick the tube to ensure that beads are covered in the buffer, and incubate on a rotator mixer at RT for 30 min. Gently flick the tube every 5 min to aid resuspension of the pellet.</li>
		<li>Pellet the beads on the magnetic rack for at least 10 min. Transfer 100 &#956;L of eluate with a P200 wide bore tip into a 0.2 mL tube.</li>
		<li>Prepare the end repair and dA-tailing reaction in a 0.2 mL tube by adding 100 &#956;L of repaired HMW DNA, 14 &#956;L of end repair/dA-tailing buffer and 7 &#956;L of end repair/dA-tailing mix. Mix the reaction by flicking gently 6 times, and spin down to remove bubbles.</li>
		<li>Incubate the reaction at 20 &#176;C for 60 min followed by 65 &#176;C for 20 min, and then hold at 22&#176;C. Transfer the sample into a new 1.5 mL tube using a P200 wide bore tip.</li>
		<li>Resuspend the magnetic beads by pipetting or vortexing. Add 48 &#956;L of beads (0.4x) to the end repair/dA-tailing reaction and mix gently by flicking the tube 6 times. Rotate the tube on a rotator mixer at RT at 20 rpm for 30 min.</li>
		<li>Repeat steps 2.1.10-2.1.13 once.</li>
		<li>Remove the tube from the magnetic rack and add 33 &#956;L of TE (10 mM Tris and 1 mM EDTA, pH 8.0). Gently flick the tube to ensure that beads are covered in the buffer, and incubate on a rotator mixer at RT for 30 min. Gently flick the tube every 5 min to aid resuspension of the pellet.</li>
		<li>Pellet the beads on the magnetic rack for at least 10 min. Transfer 30 &#956;L of eluate with a P200 wide bore tip into a new 1.5 mL tube. Take the extra 1-2 &#956;L to detect the concentration using a fluorometer. 
		NOTE: Recovery of 5-6 &#956;g at this step is expected.</li>
		<li>Prepare the ligation reaction in the 1.5 mL sample tube by adding 30 &#956;L of end-repaired HMW DNA, 20 &#956;L of adapter mix (AMX 1D), and 50 &#956;L of blunt/TA ligation master mix. Mix the reaction by flicking gently 6 times between each sequential addition and spin down to remove bubbles.</li>
		<li>Incubate the reaction at RT for 60 min.</li>
		<li>Resuspend the magnetic beads by pipetting or vortexing. Add 40 &#956;L beads (0.4x) to the ligation reaction and mix gently by flicking the tube 6 times. Rotate the tube on a rotator mixer at RT at 20 rpm for 30 min.</li>
		<li>Repeat step 2.1.10 once.</li>
		<li>Add 400 &#956;L of adapter bead binding (ABB) buffer into the tube. Flick the tube gently 6 times to resuspend the beads. Place the tube back on the magnetic rack to separate the beads from the buffer and discard supernatant.</li>
		<li>Repeat step 2.1.26 once.</li>
		<li>Spin down the sample at 1,000 x g for 2 s at RT. Place the tube back on the magnetic rack. Remove any residual buffer and air dry for 30 s. Do not over dry the pellet.</li>
		<li>Remove the tube from the magnetic rack and resuspend the pellet in 43 &#956;L of elution buffer. Gently flick the tube to ensure that beads are covered in the buffer and incubate on a rotator mixer at RT for 30 min. Gently flick the tube every 5 min to aid resuspension of the pellet.</li>
		<li>Pellet the beads on the magnetic rack for at least 10 min. Transfer 40 &#956;L of eluate with a P200 wide bore tip into a new 1.5 mL tube. Take the extra 1-2 &#956;L to detect the concentration using a fluorometer. 
		NOTE: Recovery of 1-2 &#956;g at this step is expected. The mechanical shearing-based library is ready for loading. The library can be stored on ice for up to 2 h until loading for sequencing if needed.</li>
	</ol>
	</li>
	<li>Transposase fragmentation-based library construction
	<ol>
		<li>Thaw the reagents from the transposase kit (see Table of Materials). Thaw fragmentation mix (FRA) and rapid adapter (RAP) on ice and pipette to mix. Thaw sequencing buffer (SQB), loading beads (LB), flush buffer (FLB) and flush tether (FLT) at RT and pipette to mix. Thaw loading beads (LB) at RT and pipette to mix before use. Once thawed, keep all kit components on ice. Take out the enzymes only when needed.</li>
		<li>Check the quality and quantity of the HMW DNA from step 1.21.2. Pipette out 20 &#956;L of DNA into new 1.5 mL tubes from three different locations in the HMW DNA tube using P200 wide bore tips. Take 1 &#956;L from the three aliquots to detect the concentration using a fluorometer and the quality using a UV reading. Check multiple times to confirm the results. 
		NOTE: The expected results are shown in Figure 3B. The OD260/280 value is approximately 1.9 and the OD260/230 value is approximately 2.3.</li>
		<li>Prepare the DNA tagmentation reaction in a 0.2 mL tube by adding 22 &#956;L of HMW DNA, 1 &#956;L of 10 mM Tris (pH 8.0) with 0.02% Triton X-100 and 1 &#956;L of fragmentation mix (FRA). Mix by pipetting with a P200 wide bore tip as slowly as possible 6 times, taking care not to introduce bubbles.</li>
		<li>Incubate the reaction at 30 &#176;C for 1 min followed by 80 &#176;C for 1 min, and then hold at 4 &#176;C. Transfer the mix into a new 1.5 mL tube with a P200 wide bore tip and go to next step immediately.</li>
		<li>Add 1 &#956;L of rapid adapter (RAP) to the 1.5 mL sample tube. Mix by pipetting with a P200 wide bore tip as slowly as possible 6 times, taking care not to introduce bubbles.</li>
		<li>Incubate the reaction at RT for 60 min. 
		NOTE: The transposase fragmentation-based library is ready for loading. The library can be stored on ice for up to 2 h until loading for sequencing if needed.</li>
	</ol>
	</li>
</ol>
3. Sequencing on the nanopore device
<ol>
	<li>Check the nanopore sequencing device (see Table of Materials). Make sure both the software and hardware are working and there is enough storage space.</li>
	<li>Check the flow cell. Open a new flow cell and insert the flow cell into the nanopore device. Check the box of the location the flow cell was inserted into (X1-X5). Select the correct flow cell type. Click on the Check Flow Cells workflow. Click on the Start Test button to start the flow cell QC analysis. 
	NOTE: If the reported total active pore number is less than 800, use a different new flow cell for sequencing.</li>
	<li>Prepare the priming buffer. For a mechanical shearing-based library, add 576 &#956;L of running buffer with fuel mix (RBF) and 624 &#956;L of nuclease-free water into a 1.5 mL tube. Vortex and spin down to mix the priming buffer. For a transposase fragmentation-based library, add 30 &#956;L of flush tether (FLT) to the tube of flush buffer (FLB). Vortex and spin down to mix the priming buffer.</li>
	<li>On the flow cell, move the priming port cover clockwise to expose the priming port.</li>
	<li>Set a P1000 pipette to 100 &#956;L and insert the tip into the priming port. Draw back a small volume of buffer (less than 30 &#956;L) to remove any bubbles from the flow cell. Stop pipetting once a small amount of yellow fluid enters the tip.</li>
	<li>Use a P1000 pipette to load 800 &#956;L of the priming mix into the flow cell via the priming port. To avoid introducing bubbles, add 30 &#956;L of the priming mix to cover the top of the priming port first, then insert the tip into the priming port and slowly add the rest of the priming mix. Take out the tip when there is about 50 &#956;L left. Add the rest of priming mix on the top of the priming port. The fluid will go inside by itself.</li>
	<li>Leave the setup to incubate for 5 min. In the meantime, prepare the library mix in the 1.5 mL tube containing the library. 
	NOTE: For a mechanical shearing-based library add 35 &#181;L of running buffer with fuel mix (RBF) to 40 &#181;L of the DNA library. For a transposase fragmentation-based library add 34 &#181;L of sequencing buffer (SQB) and 16 &#181;L of nuclease-free water to 25 &#181;L of the DNA library.</li>
	<li>Open the flow cell sample port cover gently to expose the sample port. Use a P1000 pipette to add 200 &#956;L of the priming mix through the priming port into the flow cell as described in step 3.5. Make sure that the priming mix is not loaded into the flow cell through the sample port.</li>
	<li>Set a P200 pipette to 80 &#956;L. Mix the library gently with a wide bore tip by pipetting up and down 6 times just prior to loading.</li>
	<li>Load the library mix dropwise through the sample port into the flow cell. Add each drop only after the previous drop is completely loaded into the port.</li>
	<li>Put back the sample port cover gently and make sure the sample port is fully covered. Move the priming port cover anticlockwise to cover the priming port. Close the device lid.</li>
	<li>Click on the New Experiment workflow. Type the library name, select the correct kit according to procedures used, and check that the settings are correct (48 h run, real-time base-calling ON).</li>
	<li>Click Start Run. After 10 min, record the flow cell ID and the active nanopore numbers (total number and each four groups&#8217; numbers) from the run information.</li>
	<li>Data analysis. Copy the data to a local computer or a cluster at any time of the sequencing or when the run is complete. Use Minimap216 (https://github.com/lh3/minimap2) to align the sequence data to the reference genome. Summarize the sequencing performance from the raw sequence data and the alignments by NanoPlot17 (https://github.com/wdecoster/NanoPlot).</li>
</ol>

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The authors declare that they have no competing financial interests.

In principle, nanopore sequencing is able to generate 100 kb to megabase reads in length11,12,13. Four major factors will affect the performance of the sequencing run and data quality: 1) active pore numbers and the activity of the pores; 2) motor protein, which controls the speed of DNA passing through the nanopore; 3) DNA template (length, purity, quality, mass); 4) sequencing adapter ligation efficiency, which determines the useable DNA from the input sample. The first two factors depend on the version of the flow cell and the sequencing kit provided by the manufacturer. The second two factors are critical steps in this protocol (HMW DNA extraction, shearing and ligation).
This protocol requires patience and practice. The quality of HMW DNA is important for ultra-long DNA libraries6. The protocol starts with cells collected with high viability (&#62;85% viable cell preferred), limiting the degraded DNA from dead cells. Any harsh process which may introduce damages to the DNA (e.g., strong disturbing, shaking, vortex, multiple pipetting, repeated freezing and thawing) should be avoided. In the design of the protocol, we omit pipetting in the entire process of DNA extraction. Wide bore tips need to be used when pipetting is necessary after the mechanical shearing during library construction and sequencing. As the nanopores are sensitive to the chemistries in the chamber buffer12, there should be as few residual contaminants (e.g., the detergents, surfactants, phenol, ethanol, proteins RNAs, etc.) as possible in the DNA. Considering the length and yield, the phenol extraction method shows the best and most reproducible results compared with multiple different extraction methods tested so far.
Despite the ability of this protocol to produce long-read sequences, several limitations still remain. First, this protocol was optimized based on the nanopore sequencing device available at time of publication; thus, it is limited to the selective nanopore-based sequencing chemistry and could be suboptimal when performed in other types of long-read sequencing devices. Second, the outcome is highly dependent on the quality of the DNA extracted from the starting material (tissues or cells). Read length will be compromised if the starting DNA is already degraded or damaged. Third, although multiple QC steps are incorporated in the protocol to check the DNA quality, the final yield and length of the reads can be affected by the flow cell and pore activity, which could be variable at this early stage of nanopore sequencing platform development.
The protocol described here uses human suspension cell line samples for DNA extraction. We have optimized the passing times in needle shearing, the ratio of HMW DNA to transposase and the ligation time to produce the described results. The protocol can be expanded in four ways. First, users can start with other cultured mammalian cells and with different amount of cells, tissues, clinical samples, or other organisms. Further optimization on lysis incubation time, reaction volume and centrifugation will be needed. Second, it is hard to predict the target size for ultra-long read sequencing. If the read lengths are shorter than expected, the users can adjust the passing times in the mechanical shearing-based method or change the ratio of the HMW DNA to transposase in the transposase fragmentation-based method. Longer binding and elution time during cleanup steps are helpful because the HMW DNA is highly viscous. Third, with different nanopore sequencing devices, one can adjust the amount and volume of the DNA to meet the criteria of the sequencer. Fourth, only those DNA ligated to sequencing adapters will be sequenced. To further improve ligation efficiency, one can attempt to titrate the adapter and ligase concentrations. Modified ligation time and molecular crowding agents such as PEG18 can be applied in future. The ultra-long DNA sequencing protocol combined with CRISPR19,20 may offer an effective tool for target enrichment sequencing.

Over the past decade, massively parallel and highly accurate second-generation high-throughput sequencing technologies have driven an explosion of biomedical discovery and technological innovation1,2,3. Despite the technical advances, the short-read data generated by the second-generation platforms are ineffective in resolving complex genomic regions and are limited in the detection of genomic structural variants (SVs), which play important roles in human evolution and diseases4,5. Furthermore, short-read data are unable to resolve repeat variation and are unsuitable for discerning haplotype phasing of genetic variants6.
Recent progress in single-molecule sequencing offers significantly longer read length, which can facilitate the detection of the full spectrum of SVs7,8,9, and offers accurate and complete assembly of complex microbial and mammalian genomes6,10. The nanopore platform performs single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores11,12,13. Unlike any existing DNA sequencing chemistry, nanopore sequencing can generate long (tens to thousands of kilobases) reads in real-time without relying on polymerase kinetics or artificial amplification of the DNA sample. Therefore, nanopore long-read sequencing (NLR-seq) holds great promise for generating ultra-long read lengths well beyond 100 kb, which would greatly advance genomic and biomedical analyses14, particularly in the low-complexity or repeat-rich regions of the genomes15.
The unique feature of nanopore sequencing is its potential to generate long reads without a theoretical length limitation. Therefore, the read length is dependent on the physical length of the DNA which is directly affected by the DNA integrity and sequencing template quality. Moreover, depending on the extent of manipulation and the number of steps involved, such as pipetting forces and extraction conditions, the quality of the DNA is highly variable. Therefore, it is challenging for one to yield long reads by just applying the standard DNA extraction protocols and manufacturer&#39;s supplied library construction methods. Toward this end, we have developed a robust method to generate ultra-long read (hundreds of kilobases) sequencing data starting from harvested cell pellets. We adopted multiple improvements in the DNA extraction and library preparation procedures. We streamlined the protocol to exclude unnecessary procedures that cause DNA degradation and damages. This protocol is composed of high molecular weight (HMW) DNA extraction, ultra-long DNA library construction, and sequencing on a nanopore platform. For a well-trained molecular biologist, it typically takes 6 h from cell harvesting to the completion of HMW DNA extraction, 90 min or 8 h for library construction depending on the shearing method, and up to a further 48 h for DNA sequencing. The use of the protocol will empower the genomics community to improve our understanding of genome complexity and gain new insight into genome variation in human diseases.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Agencourt AMPure XPbeads</td>
			<td>Beckman</td>
			<td>A63881</td>
			<td>magnetic beads for cleanup</td>
		</tr>
		<tr>
			<td>BD conventional needles</td>
			<td>Becton Dickinson</td>
			<td>305136</td>
			<td>27G, for mechanical shearing</td>
		</tr>
		<tr>
			<td>BD Luer-Lok syringe</td>
			<td>Becton Dickinson</td>
			<td>309628</td>
			<td>for mechanical shearing</td>
		</tr>
		<tr>
			<td>Blunt/TA Ligase Master Mix</td>
			<td>NEB</td>
			<td>M0367S</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10228</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>AM9261</td>
			<td>pH 8.0, 0.5 M, 500 mL</td>
		</tr>
		<tr>
			<td>Flow Cell</td>
			<td>Oxford Nanopore Technologies</td>
			<td>FLO-MIN106</td>
			<td>R9.4.1</td>
		</tr>
		<tr>
			<td>HG00773 cells</td>
			<td>Coriell Institute</td>
			<td>HG00733</td>
			<td>cells used in this protocol</td>
		</tr>
		<tr>
			<td>Ligation Sequencing Kit 1D</td>
			<td>Oxford Nanopore Technologies</td>
			<td>SQK-LSK108</td>
			<td>nanopore ligation kit</td>
		</tr>
		<tr>
			<td>MaXtract High Density tubes</td>
			<td>Qiagen</td>
			<td>129073</td>
			<td>gel tubes</td>
		</tr>
		<tr>
			<td>NEBNext FFPE DNA Repair Mix</td>
			<td>NEB</td>
			<td>M6630S</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra II End Repair/dA-Tailing Module</td>
			<td>NEB</td>
			<td>M7546S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Invitrogen</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, PBS</td>
			<td>Gibco</td>
			<td>70011044</td>
			<td>10X, pH 7.4</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:IAA</td>
			<td>Invitrogen</td>
			<td>AM9730</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Qiagen</td>
			<td>19131</td>
			<td>20 mg/mL</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32850</td>
			<td>fluorometer assays for DNA quantification</td>
		</tr>
		<tr>
			<td>Rapid Sequencing Kit</td>
			<td>Oxford Nanopore Technologies</td>
			<td>SQK-RAD004</td>
			<td>nanopore transposase kit</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Invitrogen</td>
			<td>AM9822</td>
			<td>10% (wt/vol)</td>
		</tr>
		<tr>
			<td>Sodium chloride solution</td>
			<td>Invitrogen</td>
			<td>AM9759</td>
			<td>5.0 M</td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td>Invitrogen</td>
			<td>AM9849</td>
			<td>pH 8.0</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Invitrogen</td>
			<td>AM9856</td>
			<td>pH 8.0, 1 M</td>
		</tr>
		<tr>
			<td>Triton X-100 solution</td>
			<td>Sigma-Aldrich</td>
			<td>93443</td>
			<td>~10%</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad C1000 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1851196EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>22628180</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Life Technologies</td>
			<td>AMQAF1000</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Life Technologies</td>
			<td>12321D</td>
			<td>magnetic rack</td>
		</tr>
		<tr>
			<td>Eppendorf ThermoMixer</td>
			<td>Eppendorf</td>
			<td>5382000023</td>
			<td>for incubation</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>LabRepCo</td>
			<td>LHP-5-UFMB</td>
			<td></td>
		</tr>
		<tr>
			<td>GridION</td>
			<td>Oxford Nanopore Technologies</td>
			<td>GridION X5</td>
			<td>nanopore device used in this protocol</td>
		</tr>
		<tr>
			<td>HulaMixer Sample Mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15920D</td>
			<td>rotator mixer</td>
		</tr>
		<tr>
			<td>MicroCentrifuge</td>
			<td>Benchmark Scientific</td>
			<td>C1012</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000 Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-1000</td>
			<td>for UV reading</td>
		</tr>
		<tr>
			<td>Pippin Pulse</td>
			<td>Sage Science</td>
			<td>PPI0200</td>
			<td>pulsed-field gel electrophoresis instrument</td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q33216</td>
			<td>fluorometer</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>LabRepCo</td>
			<td>LABHP-5-URBSS</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI-A236</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR</td>
			<td>89501-464</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultra-long read sequencing for whole genomic dna analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

The ultra-long DNA sequencing protocol applies HMW DNA for library construction. Therefore, it is critical to choose well-cultured cells with the live ratio &#62;85% at the cell harvesting step. The amount of cells used for DNA extraction will affect the quality and the quantity of the HMW DNA. The cell lysis does not work well if starting with too many cells. Using too few cells does not generate enough DNA for library construction because the HMW DNA precipitation is performed using gentle rotation by hand instead of high-speed centrifugation. An example of the HMW DNA after adding ice-cold 100% ethanol and rotating is shown as the white cotton-like precipitate in Figure 2.
It is important to check the quality of the input DNA before beginning the library construction. Degradation, incorrect quantification, contamination (e.g., proteins, RNAs, detergents, surfactant, and residual phenol or ethanol) and low molecular weight DNA can have a significant effect on the subsequent procedures and on the final read length. We recommend performing the QC analysis using the DNA from three different locations in the tube containing HMW DNA. From UV reading results for the HMW DNA, the OD260/OD280 value is approximately 1.9 and the OD260/OD230 value is approximately 2.3 (Figure 3A,B). These ratio values are consistent among the three tests for a good HMW DNA sample. Different shearing methods requires different volumes of input DNA. The concentration of HMW DNA needs to be &#62;200 ng/&#181;L for mechanical shearing while it needs to be &#62;1 &#181;g/&#181;L for transposase fragmentation. The concentration detected by a fluorometer is a little lower than UV reading. However, the coefficient of variation of the concentration of the same HMW DNA sample is required to be less than 15% with both the fluorometer and the UV reading assays. Mechanical shearing applies a syringe with a needle to break the HMW DNA so that the number of passes through the needle will impact the size of the sheared DNA and the final read length. It is recommended to perform size QC after needle shearing to ensure the majority of the HMW DNA is larger than 50 kb as illustrated in Figure 4. In the mechanical shearing method, 30 passes generated the best sequencing results considering both the length and output.
The N50 of a mechanical shearing-based library is 50-70 kb while a transposase fragmentation-based library is 90-100 kb. The results of four runs using the HG00733 cell line are shown in Table 1. All four runs have over 2,300 reads with length longer than 100 kb. The maximum length is longer in the transposase fragmentation-based libraries (455 kb and 489 kb) compared with the mechanical shearing-based libraries (348 kb and 387 kb) while the latter produced more total reads, indicating a higher yield. The transposase fragmentation-based library construction has fewer steps and shorter preparation time so that it will introduce fewer short fragments. The two runs using transposase have a longer mean length (&#62;30 kb) and median length (&#62;10 kb). In addition, the data shows consistent high quality in all runs (mean quality score is approximately 10.0, ~90% base accuracy). More than 97% of the total bases were aligned to the human reference genome (hg19) using Minimap216 with the default settings. The expected size distributions of the raw reads are shown in Figure 5. All runs have a large proportion of data above 50 kb while transposase fragmentation-based libraries have a higher ratio of ultra-long reads (e.g. &#62; 100 kb). This protocol has been successfully applied in multiple human cell lines (Supplementary Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58954/58954fig1.jpg" /> 
Figure 1: Schematic overview of the nanopore long-read sequencing (NLR-seq) workflow. Orange, the transposase complex. Yellow-green, the nanopore adapter. <a href="https://www.jove.com/files/ftp_upload/58954/58954fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58954/58954fig2.jpg" /> 
Figure 2: Representative DNA precipitation from phenol-chloroform extraction method. The white arrow indicates the HMW DNA. <a href="https://www.jove.com/files/ftp_upload/58954/58954fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58954/58954fig3.jpg" /> 
Figure 3: Example QC results of the HMW DNA from UV reading. (A) HMW DNA from step 1.21.1 ready for mechanical shearing-based library construction. (B) HMW DNA from step 1.21.2 for transposase fragmentation-based library construction. <a href="https://www.jove.com/files/ftp_upload/58954/58954fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58954/58954fig4.jpg" /> 
Figure 4: QC results of the needle sheared HMW DNA by pulsed-field gel electrophoresis. L1: Quick-load 1 kb DNA ladder; L2: Quick-load 1 kb extend DNA ladder. 1-8: DNA with different passing times through the needle shearing. 1-3, no shearing; 4, 10 times; 5, 20 times; 6, 30 times; 7, 40 times; 8, 50 times. This QC step is optional. <a href="https://www.jove.com/files/ftp_upload/58954/58954fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58954/58954fig5.jpg" /> 
Figure 5: Expected size distributions of the nanopore ultra-long DNA libraries. MS, mechanical shearing-based libraries. TF, transposase fragmentation-based libraries. <a href="https://www.jove.com/files/ftp_upload/58954/58954fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Mechanical shearing_rep1</td>
			<td>Mechanical shearing_rep2</td>
			<td>Transposase fragmentation_rep1</td>
			<td>Transposase fragmentation_rep2</td>
		</tr>
		<tr>
			<td>Cell line</td>
			<td>HG00733</td>
			<td>HG00733</td>
			<td>HG00733</td>
			<td>HG00733</td>
		</tr>
		<tr>
			<td>N50 of the reads</td>
			<td>55,180</td>
			<td>63,007</td>
			<td>98,237</td>
			<td>95,629</td>
		</tr>
		<tr>
			<td>Number of reads longer than 100 Kb</td>
			<td>2,500</td>
			<td>3,082</td>
			<td>2,386</td>
			<td>2,355</td>
		</tr>
		<tr>
			<td>Number of total reads</td>
			<td>97,859</td>
			<td>80,465</td>
			<td>24,166</td>
			<td>21,032</td>
		</tr>
		<tr>
			<td>Maximum length (bp)</td>
			<td>348,482</td>
			<td>387,113</td>
			<td>454,660</td>
			<td>489,426</td>
		</tr>
		<tr>
			<td>Mean length (bp)</td>
			<td>17,861</td>
			<td>20,395</td>
			<td>33,528</td>
			<td>38,175</td>
		</tr>
		<tr>
			<td>Median length (bp)</td>
			<td>5,335</td>
			<td>5,894</td>
			<td>10,249</td>
			<td>15,656</td>
		</tr>
		<tr>
			<td>Mean quality of the reads</td>
			<td>10.0</td>
			<td>10.1</td>
			<td>9.9</td>
			<td>10.0</td>
		</tr>
		<tr>
			<td>Total bases of raw reads</td>
			<td>1,747,849,822</td>
			<td>1,641,058,932</td>
			<td>810,229,733</td>
			<td>802,886,304</td>
		</tr>
		<tr>
			<td>Total bases of aligned reads</td>
			<td>1,693,300,832</td>
			<td>1,607,975,925</td>
			<td>791,422,077</td>
			<td>778,417,627</td>
		</tr>
		<tr>
			<td>Mapped ratio of total bases (hg19, Minimap2)</td>
			<td>96.9%</td>
			<td>98.0%</td>
			<td>97.7%</td>
			<td>97.0%</td>
		</tr>
		<tr>
			<td>Number of active pores</td>
			<td>1225: 480, 402, 254, 89</td>
			<td>1058: 480, 356, 176, 46</td>
			<td>958: 452, 328, 148, 30</td>
			<td>1092: 487, 367, 195, 43</td>
		</tr>
	</tbody>
</table>
Table 1: Performance metrics summary from runs with different shearing protocols.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Library 1</td>
			<td>Library 2</td>
		</tr>
		<tr>
			<td>Cell line</td>
			<td>K562</td>
			<td>GM19240</td>
		</tr>
		<tr>
			<td>Cell Ordering Information</td>
			<td>ATCC, cat. No. CCL-243</td>
			<td>Coriell Institute, cat. No. GM19240</td>
		</tr>
		<tr>
			<td>Protocol</td>
			<td>mechanical shearing</td>
			<td>mechanical shearing</td>
		</tr>
		<tr>
			<td>N50 of the reads</td>
			<td>60,063</td>
			<td>55,295</td>
		</tr>
		<tr>
			<td>Number of total reads</td>
			<td>193,783</td>
			<td>120,807</td>
		</tr>
		<tr>
			<td>Median length (bp)</td>
			<td>1,843</td>
			<td>4,688</td>
		</tr>
		<tr>
			<td>Mean length (bp)</td>
			<td>9,825</td>
			<td>17,408</td>
		</tr>
		<tr>
			<td>Maximum length (bp)</td>
			<td>548,780</td>
			<td>212,338</td>
		</tr>
		<tr>
			<td>Total bases of raw reads</td>
			<td>1,903,989,686</td>
			<td>2,103,015,331</td>
		</tr>
		<tr>
			<td>Total bases of aligned reads</td>
			<td>1,837,350,047</td>
			<td>1,997,419,761</td>
		</tr>
		<tr>
			<td>Mapped ratio of total bases (hg19, Minimap2)</td>
			<td>96.6%</td>
			<td>95.0%</td>
		</tr>
		<tr>
			<td>Number of active pores</td>
			<td>1111: 482, 371, 203, 55</td>
			<td>1032: 447, 333, 196, 56</td>
		</tr>
	</tbody>
</table>
Supplementary Table 1: Summary of two NLR-seq runs using other cell lines with the mechanical shearing protocol.

Watch this Scientific Journal Video about Ultra-long Read Sequencing for Whole Genomic DNA Analysis at JoVE.com

Ultra-long Read Sequencing for Whole Genomic DNA Analysis | Bioengineering | Video

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

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22.4K Views.  Jackson Laboratory for Genomic Medicine. Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Video: Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...