Ultra-long Read Sequencing for Whole Genomic DNA Analysis

This protocol fills a technical gap in our ability to generate ultra-long sequence arrays. It enables us to examine genome complexity that is limited by current short-read approaches in genomics. The method is unique in performance, robustness, versatility, and potential to integrate with promising new applications.

It is applicable to different materials and can be modulated for different instant sizes. This technique has begun revolutionizing the clinical landscape. Its diagnosis utility in repeat expansion disorders has overcome many current shortcomings.

Some transplant registries have utilized high-resolution HLA-typing for better clinical outcome. These methods provide previously inaccessible long-range information such as phasing and complex structural variance. Coupled with epigenetic readout in a single test, it will enable precision medicine.

It's a long protocol with many new techniques and it's hard to know if it's working until final sequencing. I recommend practicing loading on old flow cells before running samples. Collaborators have said some techniques are confusing compared to short-read approaches.

Specifically, the extraction is gentler due to the high quality, high-molecular weight DNA. To begin the extraction of HMW DNA, first add 200 microliters of the cell suspension to 10 milliliters of lysis buffer in a 50 milliliter tube. Then, vortex at the highest speed for three seconds.

And incubate the solution at 37 degrees celsius for one hour. At the end of the incubation, add two microliters of RNS-A to the lysate. Gently rotate the 50 milliliter tube to mix the sample and incubate at 37 degrees celsius for one hour.

After incubation, add 10 milliliters of the phenol layer of phenol-chloroform-isoamyl alcohol mixture to the lysate. And put the tube on a rotator at 20 rpm for 10 minutes under a fume hood. After centrifugation with gel tubes, carefully pour the supernatant into a new 50 milliliter tube.

Then, add 25 milliliters of ice-cold 100%ethanol, and gently rotate the tube by hand, until the DNA precipitates. Bend a 20 microliter tip to make a hook. Using the hook, carefully take out the HMW DNA and let the liquid drop off.

Then, place the DNA into a 50 milliliter tube containing 40 milliliters of 70%ethanol, and gently invert the tube three times to wash the DNA. To prepare the mechanical shearing-based library, first use a 100 milliliter needle-free syringe to aspire all the DNA. Then, put a 27 gauge needle onto the syringe and eject all the DNA into the dish gently and slowly.

Repeat 29 times. Next, add 143 microliters of the re-suspended magnetic beads and 143 microliters of the DNA repair reaction mixture to a tube. Flick the tube six times to mix gently, and put the tube on a rotator at 20 rpm for 30 minutes.

Centrifuge the tube at 1000G for two seconds at room temperature to spin down the sample, and then place the tube on a magnetic rack for 10 minutes. Next, discard the supernatant while the tube is still on the rack. Add 400 microliters of the freshly-prepared 70%ethanol.

Wait for 30 seconds. And then remove the ethanol. Then, centrifuge the tube at 1000G for two seconds at room temperature to spin down the sample.

Place the tube back on the magnetic rack and remove any residual ethanol. Air-dry the pellet for 30 seconds. Next, remove the tube from the magnetic rack and add 103 microliters of T-E buffer.

Then, flick the tube gently to ensure that beads are covered in the buffer and put the tube on the rotator at room temperature for 30 minutes. Finally, place the tube back on the magnetic rack for 10 minutes to pellet the beads. Use a P200 wide bore tip to transfer 100 microliters of the eluate to a 0.2 milliliter tube, and proceed to end-repair dA-tailing and adapter ligation reactions.

To prepare the transposase fragmentation-based library, first make a DNA tagmentation reaction by adding 22 microliters of the HMW DNA, one microliter of 10 millimolar Triss, pH 8, with 0.02%Triton X-100, and 1 microliter of the fragmentation mix, to a 0.2 milliliter tube. Next, use a P200 wide bore tip to mix the reaction six times, and incubate at 30 degrees celsius for one minute, followed by 80 degrees celsius for one minute. Hold at four degrees celsius.

Finally, use a P200 wide bore tip to transfer the reaction to a 1.5 milliliter tube and quickly add 1 microliter of rapid-adapter mix. Use a P200 wide bore tip to mix the reaction six times. Incubate the reaction at room temperature for one hour, and proceed to sequencing.

To begin sequencing, first insert a new flow cell into one of the channels of the Nanopore device. Then, on the accompanying sequencing control software, check the location box of the flow cell. Select the correct flow cell type and click on the check flow cell's workflow.

Click on the start test button to start the flow cell QC analysis. Next, with a P1000 pipette set to 100 microliters, draw back less than 30 microliters of buffer to remove bubbles from the flow cell. Add 30 microliters of the priming mix to cover the top of the priming port to avoid introducing bubbles.

Then, pipette 800 microliters of the priming mix into the priming port to load the flow cell. Take out the tip when there is about 50 microliters of the priming mix left, and add the rest of the priming mix on the top of the priming port. Next, with a P1000 pipette, add 200 microliters of the priming mix into the flow cell.

Then, just prior to loading, use a P200 pipette set to 80 microliters to pipette up and down the DNA library six times. Finally, load the library mix drop-wise into the flow cell through the sample port, and proceed to sequencing and the data analysis. QC analysis of DNA ready for mechanical shearing-based library construction resulted in the 260 to 280 purity ratio of approximately 1.9, and the 260 to 230 ratio of approximately 2.3, showing a good DNA sample.

The same result was seen using DNA ready-for-transposase fragmentation-based library construction. Size quality control of the needle-sheared HMW DNA by pulse field gel electrophoresis showed that the majority of the HMW DNA was larger than 50 kilobases. The results of four runs using the HG00733 cell showed that the N50 of a mechanical shearing-based library was shorter than a transposase fragmentation-based library.

Also, all four runs had over 2300 reads with length longer than 100 kilobases. The maximum length was longer in the transposase fragmentation-based libraries with its shorter preparation time, compared to the mechanical shearing-based libraries. The mechanical shearing-based libraries produced more total reads compared to the transposase fragmentation-based libraries, indicating a higher yield.

Both libraries showed consistent high quality and more than 97%of their total bases were aligned with the human reference genome. The expected size distributions showed that all four runs had a large proportion of data above 50 kilobases, while transposase fragmentation-based libraries had a higher ratio of ultra-long reads. The overall goal is to keep the DNA intact, so it's important to avoid any harsh handling of the DNA.

A key factor that can maximize the value of the method is the computational analysis. Our team has developed a long-read analysis pipeline called PICKY that can detect a full range of SVs with high sensitivity. It will open new avenues to exciting areas like detecting complex SVs and phasing rearrangements and modifications at the single molecule level.

This will greatly improve our understanding of genome architecture. Phenol is very corrosive. You must work with it in a fume hood with personal protective equipment, including gloves, safety glasses, a lab coat, long pants and shoes.