Third generation nanopore sequencing is a powerful novel sequencing technology that allows to very easily obtain sequence information from a wide variety of samples. The main advantages of this technique are the portability of the sequencing device, the extremely long read length, and the real-time availability of data. Nanopore sequencing is a particularly helpful technology during disease outbreaks in remote locations.
It has successfully been used in field laboratories during and after Ebola virus outbreaks in West and Central Africa. Via nanospore sequencing and the use of the MinION device for this purpose is rather easy, care must be taken during library preparation and during flow cell loading. To begin this procedure, set up a touchdown PCR to amplify the cDNA with primer set one using a hot start high fidelity DNA polymerase with the appropriate reaction buffer in a 50 microliter reaction volume with one microliter template.
If possible, set up the reaction on ice or in a cool block at four degrees Celsius. Incubate the reaction in a thermocycler as outlined in the text protocol. After this, transfer 50 microliters of the PCR product into a 1.5 milliliter DNA low binding reaction tube.
Resuspend the magnetic beads thoroughly by vortexing. Then add 50 microliters of beads to the PCR reaction and mix well. Incubate the sample on a rotating mixer for five minutes at room temperature while rotating at 15 RPM.
Briefly spin down the sample and then place the tube on a magnetic rack to pellet the magnetic beads. Wait until the supernatant has been completely clarified before continuing. Next, aspirate the supernatant without disturbing the bead pellet and discard it.
Pipette 200 microliters of 70%ethanol into the reaction tube and incubate for 30 seconds at room temperature. Aspirate the ethanol without disturbing the bead pellet and discard it. Repeat this washing process with ethanol once more for a total of two washes.
Air dry the pellet for one minute at room temperature. Then remove the reaction tube from the magnetic rack. Resuspend the pellet in 30 microliters of nuclease free water and incubate at room temperature for two minutes.
Place the reaction tube back on the magnetic rack and wait until the reaction beads are completely pelleted. After this, remove the supernatant without disturbing the pellet and transfer to a new 1.5 milliliter reaction tube. If several samples are to be sequenced at the same time, barcodes can now be added by two additional PCR steps followed by DNA cleanup as previously shown.
First, combine an equal amount of barcoded DNA from each sample for a total of one microgram of DNA in a volume of 45 microliters in a 0.2 milliliter reaction tube. For dA-Tailing, add seven microliters of end prep reaction buffer, three microliters of end prep enzyme mix, and five microliters of nuclease free water. Gently flick the tube to mix.
In a thermocycler, incubate the reaction for five minutes at 20 degrees Celsius followed by five minutes at 65 degrees Celsius. Next, perform PCR purification of the reaction product as outlined in the text protocol. Optionally, take one microliter to quantify the concentration of the sample using a UV spectrophotometer.
Combine 22.5 microliters of the previously obtained purified DNA with 2.5 microliters of 1D2 adapter and 25 microliters of Blunt/TA Ligase Master Mix in a new 1.5 milliliter DNA low binding reaction tube. Gently flick the tube to mix. Briefly spin down the mixture and incubate at room temperature for 10 minutes.
Then perform PCR purification of the reaction product as outlined in the text protocol. Combine 45 microliters of the reaction product with five microliters of barcode adapter mix and 50 microliters of Blunt/TA Ligase Master Mix in a DNA low binding reaction tube. Gently flick to mix and incubate for 10 minutes at room temperature.
Purify the reaction product as described in the text protocol and store the resulting product on ice or at four degrees Celsius until ready to use. To begin, perform a quality check on the flow cell before use. To this end, connect the sequencing device to the host computer and open the software.
Insert a flow cell into the sequencing device. Then choose the flow cell type from the selector box and click available to confirm. At the bottom of the screen, click check flow cell and choose the correct flow cell type.
Click start test to start the quality check. A minimum of 800 active nanopores in total is required for the flow cell to be usable. First, open the priming port cover by sliding it in a clockwise direction.
Set a P1000 pipette to 200 microliters and insert the tip into the priming port. Then adjust the pipette to 230 microliters while keeping the tip in the priming port to draw up 20 to 30 microliters of buffer and remove any air bubbles. In a new 1.5 milliliter DNA low binding reaction tube, prepare the priming mix by combining 576 microliters of RBF buffer with 624 microliters of nuclease free water.
Carefully pipette 800 microliters of the prepared priming mix into the priming port and wait five minutes. After this, lift the sample port cover and pipette an additional 200 microliters of the prepared priming mix into the priming port. Pipette 35 microliters of the RBF buffer into a new clean 1.5 milliliter DNA low binding reaction tube.
Thoroughly mix LLB beads by pipetting and add 25.5 microliters of the beads to the RBF buffer. Next, add 2.5 microliters of nuclease free water and 12 microliters of DNA library and mix by pipetting. Add 75 microliters of the sample mixture in a slow drop wise fashion to the flow cell via the sample port.
Then replace the sample port cover, close the priming port and close the lid of the sequencing device. Within the software, confirm that the flow cell is still available. Open a new experiment and set up the run parameters by selecting the kit used.
Select live basecalling. Start the sequencing run by clicking begin experiment. Continue the sequencing run until sufficient experimental data is collected.
In a representative experiment, the RNA is extracted from 10 different blood samples from five animal species. RNA concentrations are observed between 43 nanograms and 543 nanograms per microliter. After amplification by RT-PCR, gel analysis of the MPC1 PCR products shows various outcomes with markedly weaker bands for samples BC01 and BC02.
These differences are most likely caused by differences in sample quality although differences in PCR efficacy due to differences in primer binding to the MCP1 gene of different species cannot be excluded. However, these differences in yield and/or amplification efficiency do not markedly impact the overall sequencing outcome. A subset of 10, 000 obtained reads is then selected and demultiplexed for further analysis.
Of the 10, 000 reads analyzed, 5, 457 show a length between 1, 750 and 2, 000 nucleotides which matches the expected sizes for the PCR fragments amplified as part of our workflow. An additional peak in the length distribution of reads is observed between 250 to 500 nucleotides which can be attributed to unspecific PCR products. Demultiplexing of reads allow the assignment of 87.6%of the reads to one of the 10 samples analyzed.
While this method is quite reliable, under field conditions, the PCR amplification is the most problematic step. During our experiences, nested protocols were often necessary to amplify target sequences. Besides sequencing animal host genes, this method can also be used to sequence any DNA or RNA including viral or bacterial pathogens.
Nanopore sequencing is therefore now increasingly used not only during disease outbreaks or for scientific studies in remote locations but also in regular laboratories where the long read lengths open up exciting new research possibilities. While none of the used reagents are particularly hazardous, standard good laboratory practices should be followed. Obviously when sequencing disease agents, all necessary precautions for handling these agents must be observed.
