The authors thank Allison Groseth for critical reading of the manuscript. This work was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany through the Federal Office for Agriculture and Food (BLE).

Samples were collected following the Njala University Institutional Review Board (NUIRB) protocol no. IRB00008861/FWA00018924.
1. RNA Extraction from Blood Samples
<ol>
	<li>Collect 3 mL of whole blood from the species to be analyzed into a blood collection tube prefilled with 6 mL of DNA/RNA stabilizing reagent (see Table of Materials) and mix by inverting 5 times. Store the blood sample for up to one month at 4 &#176;C.</li>
	<li>Transfer the contents of the blood collection tube into a 50 mL collection tube, add 120 &#181;L of proteinase K, and mix by vortexing for 5 s. Incubate the sample for 30 min at room temperature.</li>
	<li>Add 9 mL of isopropanol to the mixture and vortex for 5 s.</li>
	<li>Place a reservoir onto an RNA purification spin column (see Table of Materials) and place the assembly onto a vacuum manifold (see Table of Materials). Add the sample mixture into the reservoir. Apply a vacuum until all liquid has passed through the column.</li>
	<li>Alternatively, if no vacuum manifold is available, the blood can be passed through the column in 700 &#181;L portions by repeated centrifugation for 30 s at 12,000 x g with the flow-through discarded between centrifugation steps. However, this will require approximately 26 centrifugation steps.</li>
	<li>Place the RNA purification spin column into a collection tube and add 400 &#181;L DNA/RNA prep buffer (see Table of Materials). Centrifuge at 12,000 x g for 30 s and discard the flow-through.</li>
	<li>Add 400 &#181;L of DNA/RNA wash buffer (see Table of Materials) to the column, centrifuge at 12,000 x g for 30 s and discard the flow-through.</li>
	<li>Mix 5 &#181;L of DNase I (1 U/&#181;L) (see Table of Materials) with 75 &#181;L of DNA digestion buffer (see Table of Materials) and add the mixture onto the column. Incubate for 15 min at room temperature.</li>
	<li>Add 400 &#181;L of DNA/RNA prep buffer to the column and centrifuge at 12,000 x g for 30 s. Discard the flow-through.</li>
	<li>Wash the column with 700 &#181;L of DNA/RNA wash buffer and centrifuge at 12,000 x g for 30 s. Discard the flow-through.</li>
	<li>Repeat step 1.9 with 400 &#181;L of DNA/RNA wash buffer and centrifuge at 12,000 x g for 2 min to remove all residual wash buffer and to dry the column. When removing the column from the collection tube, make sure not to wet the underside of the column with the buffer in the collection tube.</li>
	<li>Place the column into a new 1.5 mL microcentrifuge tube and add 70 &#181;L of nuclease-free water. Incubate for 1 min at room temperature and centrifuge for 30 s at 12,000 x g. Store the RNA at -80 &#176;C until further use (or use immediately).
	<ol>
		<li>OPTIONAL: To quantify the RNA, take an aliquot and determine the concentration using a UV spectrophotometer (see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Reverse Transcription of NPC1 mRNA into cDNA
<ol>
	<li>In a 0.2 mL reaction tube, add 8 &#181;L of template RNA (1 pg to 2.5 &#181;g of RNA) and 1 &#181;L each of 10x DNase buffer and DNase enzyme (see Table of Materials). Incubate at 37 &#176;C for 2 min. Subsequently, centrifuge the reaction briefly and place it on ice.</li>
	<li>Add 4 &#181;L of reverse transcriptase master mix (see Table of Materials) and 6 &#181;L of nuclease-free water to the reaction tube and mix gently. Incubate the reaction in a thermocycler for 10 min at 25 &#176;C (for primer annealing), followed by 10 min at 50 &#176;C (for reverse transcription of RNA). To inactivate the enzyme, incubate for 5 min at 85 &#176;C.</li>
	<li>Transfer cDNA to a new 1.5 mL microcentrifuge tube and store at -80 &#176;C until further use (or use immediately).</li>
</ol>
3. Amplification of the NPC1 Open Reading Frame
<ol>
	<li>Initial amplification step
	<ol>
		<li>Set up a touchdown PCR15,16 to amplify the NPC1 cDNA with Primer Set 1 (see Table 1), using a hot start high fidelity DNA polymerase (see Table of Materials) with the appropriate reaction buffer in a 50 &#181;L reaction volume with 1 &#181;L template. If possible, set up the reaction on ice or in a 4 &#176;C cool block.</li>
		<li>Incubate the reaction in a thermocycler with an initial denaturation step of 30 s at 98 &#176;C, followed by 10 cycles with denaturation at 98 &#176;C for 10 s, primer annealing for 20 s at 65 &#176;C, lowering the temperature by 0.5 &#176;C per cycle, and elongation for 1 min at 72 &#176;C. Subsequently, run an additional 20 cycles with 10 s at 98 &#176;C, 20 s at 60 &#176;C, and 1 min at 72 &#176;C, followed by a final elongation step of 5 min at 72 &#176;C.</li>
	</ol>
	</li>
	<li>PCR purification using magnetic beads
	<ol>
		<li>Transfer 50 &#181;L of PCR product into a 1.5 mL DNA-low binding reaction tube (see Table of Materials). Resuspend magnetic beads (see Table of Materials) thoroughly by vortexing and add 50 &#181;L beads to the PCR reaction. Mix well. Incubate the sample on a rotating mixer (see Table of Materials) for 5 min at room temperature (at 15 rpm).</li>
		<li>Briefly spin down the sample and place the 1.5 mL microcentrifuge tube on a magnetic rack (see Table of Materials) to pellet the magnetic beads. Wait until the supernatant has been completely clarified before continuing with the next step.</li>
		<li>Aspirate the supernatant without disturbing the bead pellet and discard.</li>
		<li>Pipette 200 &#181;L of 70% ethanol into the reaction tube and incubate for 30 s. Aspirate the ethanol without disturbing the pellet and discard. Repeat for a total of two washes. Make sure that no ethanol is left. It may be necessary to first aspirate with a larger pipette (e.g., 1000 &#181;L), and then to remove any remaining ethanol droplets with a smaller pipette (e.g., 10 &#181;L).</li>
		<li>Air-dry the pellet for 1 min at room temperature.</li>
		<li>Remove the reaction tube from the magnetic rack, resuspend the pellet in 30 &#181;L of nuclease-free water and incubate for 2 min at room temperature.</li>
		<li>Place the reaction tube back on the magnetic rack and wait until the beads are completely pelleted.</li>
		<li>Remove the supernatant without disturbing the pellet and transfer it to a new 1.5 mL reaction tube.</li>
	</ol>
	</li>
	<li>Addition of barcode adapters by nested PCR
	<ol>
		<li>Set up a 50 &#181;L touchdown PCR with hot start High-Fidelity DNA polymerase with 5x reaction buffer and Primer Set 2 (see Table 1). Primers in this set consist of a target sequence-specific region to allow binding of PCR products generated in step 3.2 (inside of the primer set 1 sequences), as well as an adapter sequence that is used as target in the subsequent barcoding PCR reaction (cf. section 4). If possible, set up the reaction on ice or in a 4 &#176;C cool block. Use 1 &#181;L of the purified PCR product prepared in section 3.2 as template.</li>
		<li>Incubate the reaction mix in a thermocycler using an initial denaturation step of 30 s at 98 &#176;C, followed by 10 cycles with denaturation at 98 &#176;C for 10 s, primer annealing for 20 s at 65 &#176;C, lowering the temperature by 0.5 &#176;C per cycle, and elongation for 1 min at 72 &#176;C. Subsequently, incubate the reaction for a further 30 cycles for 10 s at 98 &#176;C, 20 s at 71 &#176;C, and 1 min at 72 &#176;C, followed by a final elongation step of 5 min at 72 &#176;C.</li>
		<li>Clean up the PCR product using magnetic beads as described in section 3.2.</li>
	</ol>
	</li>
</ol>
4. Barcoding of NPC1 Amplicons
<ol>
	<li>For each PCR product generated in section 3, set up a barcoding PCR reaction in a 0.2 mL reaction tube using 50 &#181;L of Taq DNA polymerase 2x master mix (see Table of Materials), 2 &#181;L of one of the barcode primers from a PCR barcoding kit (see Table of Materials and Table 2), and 1 &#181;L of purified PCR product from step 3.3.2 as template. Add 47 &#181;L of nuclease free water to obtain a final volume of 100 &#181;L.</li>
	<li>Incubate the reaction in a thermocycler at 95 &#176;C for 3 min as an initial denaturation. Subsequently, run 15 cycles for 15 s at 95 &#176;C, 15 s at 62 &#176;C, and 1.5 min at 65 &#176;C. As a final elongation, incubate the reaction at 65 &#176;C for 5 min.</li>
	<li>Purify the PCR product as described under 3.2, but use 100 &#181;L of magnetic beads and elute in 30 &#181;L of nuclease-free water.</li>
	<li>If possible, quantify the sample using a UV spectrophotometer.</li>
</ol>
5. Library Preparation
<ol>
	<li>Combine an equal amount of barcoded DNA from each sample for a total of 1 &#181;g of DNA in a volume of 45 &#181;L (if necessary, add nuclease-free water) in a 0.2 mL reaction tube. If no UV spectrophotometer is available, use equal volumes of each sample. For dA-tailing, add 7 &#181;L of End-prep reaction buffer (see Table of Materials), 3 &#181;L of End-prep enzyme mix (see Table of Materials), and 5 &#181;L of nuclease-free water. Mix gently by flicking the tube.</li>
	<li>Incubate the reaction for 5 min at 20 &#176;C, followed by 5 min at 65 &#176;C in a thermocycler.</li>
	<li>Purify the reaction product as described in section 3.2, but use 60 &#181;L of magnetic beads and elute in 25 &#181;L of nuclease-free water.</li>
	<li>OPTIONAL: Take 1 &#181;L to quantify the concentration of the sample using a UV spectrophotometer. The total amount should be more than 700 ng.</li>
	<li>Combine 22.5 &#181;L of purified DNA from step 5.3 with 2.5 &#181;L of 1D2 adapter (see Table of Materials) and 25 &#181;L of blunt/TA ligase master mix (see Table of Materials) in a new 1.5 mL DNA-low binding reaction tube, mix gently by flicking, and briefly spin down. Incubate for 10 min at room temperature.</li>
	<li>Purify the reaction product as described in section 3.2, but use 20 &#181;L of magnetic beads, increase the incubation time for DNA binding to 10 min, perform two wash steps with 1 mL of ethanol each, and elute in 46 &#181;L of nuclease-free water.</li>
	<li>Combine 45 &#181;L of the reaction product from step 5.6 with 5 &#181;L of barcode adapter mix (see Table of Materials) and 50 &#181;L of blunt/TA ligase master mix in a DNA-low binding reaction tube. Mix gently by flicking and incubate for 10 min at room temperature.</li>
	<li>Purify the reaction product as described in section 3.2, but use 40 &#181;L of magnetic beads, do two wash steps with 140 &#181;L of ABB buffer (see Table of Materials) instead of ethanol, resuspend the beads by flicking and pellet on a magnetic rack. Elute in 15 &#181;L of elution buffer (see Table of Materials). Increase the incubation times for the initial binding of DNA to the beads as well as for the elution step to 10 min. Store the resulting product on ice or at 4 &#176;C until use.</li>
</ol>
6. Quality Check of Flow Cell
<ol>
	<li>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.</li>
	<li>Insert a flow cell (see Table of Materials) into the sequencing device and choose the flow cell type from the selector box and confirm by clicking Available.</li>
	<li>Click Check flow cell in the bottom of the screen and choose the correct flow cell type.</li>
	<li>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.</li>
</ol>
7. Loading the Flow Cell and Starting the Sequencing Run
<ol>
	<li>Open the priming port cover by sliding it in a clockwise direction. Set a P1000 pipette to 200 &#181;L and insert the tip into the priming port. Adjust the pipette to 230 &#181;L while keeping the tip in the priming port, to draw up 20-30 &#181;L buffer and remove any air bubbles.</li>
	<li>In a new 1.5 mL DNA-low binding reaction tube prepare the priming mix by combining 576 &#181;L of RBF buffer (see Table of Materials) with 624 &#181;L of nuclease-free water.</li>
	<li>Carefully pipette 800 &#181;L of the prepared priming mix into the priming port and wait 5 min. Lift the sample port cover, and pipette an additional 200 &#181;L of the prepared priming mix into the priming port.</li>
	<li>Pipette 35 &#181;L of RBF buffer into a new, clean 1.5 mL DNA low-binding reaction tube. Thoroughly mix LLB beads (see Table of Materials) by pipetting and add 25.5 &#181;L of the beads to the RBF buffer. Add 2.5 &#181;L nuclease-free water and 12 &#181;L of DNA library from step 5.8 and mix by pipetting.</li>
	<li>Add 75 &#181;L of the sample mixture in a slow dropwise fashion to the flow cell via the sample port.</li>
	<li>Replace the sample port cover, close the priming port, and close the lid of the sequencing device.</li>
	<li>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 base-calling. Start the sequencing run by clicking Begin Experiment. Continue the sequencing run until sufficient experimental data is collected.</li>
</ol>

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TH participated in the Oxford Nanopore Technologies (ONT) MinION early access program from 2014 to 2015, and received MinION devices and flow cells for a previous study 7 performed by the National Institutes of Health, USA, free of charge or at reduced costs. He was invited by ONT to present part of that work at the London Calling 2015 meeting in London, UK, and ONT payed for transportation and accommodation. For the work presented in this manuscript, no benefits (e.g. hardware or reagents at reduced cost, travel reimbursements etc.) were obtained from ONT. AM, KF and RS have nothing to disclose.

Over the last two decades, sequencing of biological samples has become an increasingly important aspect of studies in a wide range of subject areas. The development of second-generation sequencing systems based on the sequencing of a dense array of DNA features using iterative cycles of enzymatic manipulation and image-based data acquisition1 has dramatically increased throughput compared to the traditional Sanger sequencing technique, and allows analysis of multiple samples as well as various nucleic acid species in a given sample in parallel4. However, for most of the commonly used second-generation systems, only short reads are produced, and all platforms rely on sensitive, bulky, and expensive equipment3,4.
In contrast to second-generation sequencing platforms, the sequencing device used in this protocol is based on nanopore technology. Here a single-stranded nucleic acid molecule passes through a nanopore, resulting in modulation of an ionic current that is also flowing through the same nanopore, and which can be measured and back-translated to infer the sequence of the nucleic acid molecule. This third-generation sequencing approach imparts a number of advantages over other approaches. The main advantages that are directly related to the unique working principle of this technology are the extremely long read length produced (read lengths of up to 8.8 x 105 nucleotides have been reported6), the ability to sequence not only DNA but also RNA directly, which was recently demonstrated for a complete influenza virus genome17, and the ability to analyze data in real-time as they are being generated, which allows rapid metagenomics detection of pathogens within minutes18. Additional practical advantages are the extremely small size of the nanopore sequencing device, allowing its use in any laboratory or on field missions to remote locations19,20, and the low price in comparison to other sequencing platforms. In terms of running costs, currently a new flow cell is required for each sequencing run, which results in costs of about $1,100 per run for the flow cell and library preparation reagents. These costs can be reduced in some cases by washing and reusing the flow cell, or by barcoding and sequencing multiple samples in a single run. Also, a novel type of flow cell is currently being beta-tested by a small number of laboratories, which will require the use of a flow cell adaptor (called a &#8220;flongle&#8221;), and should significantly reduce flow cell price and thus running costs.
The major shortcoming of nanopore sequencing remains its accuracy, with single read accuracies in the range of 83 to 86% being reported6,21,22, and most of the inaccuracies being caused by insertion/deletions (indels)5,21. However, high read depth can compensate for these inaccuracies, and a recent study suggested based on theoretical considerations that a read depth of &#62;10 might increase overall accuracy to &#62;99.8%21. Nevertheless, further improvements in accuracy will be needed, particularly if analysis is to be performed on a single molecule level rather than on a consensus sequence level. The use of 1D2 technology as described in this protocol, which is based on the addition of the 1D2 and barcode adapters (cf. section 5.5) that result in both strands of a single DNA molecule being sequenced by the same nanopore, increases read accuracy since information from both DNA strands can be used for sequence determination. Further, a workaround strategy that can be pursued in order to combine the advantages of nanopore sequencing (particularly long read length) with the higher accuracy of other sequencing technologies is to use nanopore sequencing information as a scaffold, which is then polished using sequencing data from other platforms6.
The most critical factor for the success of the protocol presented here is sample quality, and particularly the amount and quality of the extracted RNA. Proper storage and prompt extraction of the RNA help in achieving an adequate RNA yield. The use of appropriate blood collection tubes allows the storage of blood samples for up to one month, but blood clotting can be an issue, particularly when samples are being stored at elevated temperatures, which can be the case under field conditions. The second critical step is the amplification of target sequences, and particular under field conditions PCR reactions often perform less well than under standard laboratory conditions7. To this end, careful primer design and optimization is paramount to achieve robust amplification. Additionally, nested PCR approaches and touchdown PCR, as used in this protocol, can increase both specificity and sensitivity of target gene amplification4,7. Indeed, in our experience in Liberia and Guinea with this technology nested protocols were required under field conditions with field samples even for primer sets which allowed amplification of targets from laboratory samples and under laboratory conditions with a single round of PCR (7 and unpublished results).
In contrast to these more critical steps, library preparation and the sequencing run itself are rather robust procedures. However, under field conditions practical issues such as the availability of certain pieces of equipment can be problematic. For example, a UV spectrophotometer is needed to determine DNA concentrations prior to library preparation of barcoded samples. However, should such a device not be available under field conditions, an equal volume of each sample can simply be combined to make up the 45 &#181;L required for library preparation, with differences in sample input material then usually being mitigated by the large number of reads. Similarly, the need for internet connectivity for the sequencing run can be an issue, even though the base-calling no longer has to be performed online but can be done locally; however, this necessity can be removed under certain circumstances by the manufacturer if required.
In summary, the presented protocol allows relatively low-cost sequencing in locations with no access to traditional sequencing equipment, including in remote locations. It can easily be adapted to any target RNA or DNA, thus allowing researchers to answer numerous biological questions.

Sequencing is a powerful and important tool in biological and biomedical research. It allows analysis of genomes, genetic variations, and RNA expression profiles, and thus plays an important role in the investigation of human and animal diseases alike1,2. Sanger sequencing, one of the oldest methods available for DNA sequencing, is still routinely used to this day and has been a corner-stone of molecular biology. Over the past 50 years, this technology has been improved to achieve read-lengths of more than 1,000 nt and an accuracy as high as 99.999%1. However, Sanger sequencing also has limitations. Sequencing a larger set of samples or the analysis of whole genomes with this method is time consuming and expensive1,3. Second-generation (next-generation) DNA sequencing methods such as 454 pyrosequencing and Illumina technology have allowed us to significantly reduce the cost and workload required for sequencing in the last decade, and have led to a tremendous increase in the amount of biological sequence information available4. Nevertheless, individual sequencing runs using these second-generation technologies are expensive, and sequencing under field conditions is challenging, as the necessary equipment is bulky and fragile (similar to Sanger sequencing devices), and often has to be calibrated and serviced by specially trained personnel. Also, for many of the second-generation technologies read-lengths are rather limited, which often makes downstream bioinformatics analysis of these data challenging.
Third-generation sequencing using pocket-sized nanopore sequencing devices (see Table of Materials) can serve as an alternative to these established sequencing platforms. In these devices a single-stranded DNA or RNA molecule passes through a nanopore simultaneously with an ionic current that is then measured by a sensor (Figure 1). As the strand traverses the nanopore, the modulation of the current by the nucleotides present in the pore at any given time is detected, and computationally back-translated into the nucleotide sequence5. Because of this operational principle, nanopore sequencing allows both the generation of very long reads (close to 1 x 106 nucleotides6) and the analysis of sequencing data in real time. Barcoding is possible by attaching defined nucleotide sequences to the nucleic acids in a sample, which allows analysis of multiple samples in a single sequencing run, thus increasing sample throughput and lowering per sample costs. Due to their high portability and ease of use, nanopore sequencing devices have been used successfully in the field during the recent Ebola virus disease epidemic in West Africa, highlighting their suitability for rapid deployment into remote regions7,8.
Here, we describe a detailed field-compatible protocol for the sequencing of mRNA encoding for the Niemann-Pick C1 (NPC1) protein, which is the obligate entry receptor for filoviruses such as ebolaviruses, and has been shown to limit species susceptibility to these viruses9,10. The protocol encompasses extraction of whole RNA from blood samples, specific amplification of NPC1 mRNA by RT-PCR, barcoding of samples, library preparation and sequencing with a nanopore sequencing device. Data analysis cannot be discussed due to space limitations, although some basic directions are provided in the representative results; however, the interested reader is referred to a previous publication11 for a more detailed description of the workflow we used, as well as to publications by others12,13,14 for detailed information regarding the analysis tools used in this workflow.

<table><tbody><tr><td>1D2 adapter, barcode adapter mix, ABB buffer, elution buffer, RBF buffer, LBB beads</td><td>Oxford Nanopore Technologies</td><td>SQK-LSK308</td><td>1D&amp;sup2; Sequencing Kit</td></tr><tr><td>Blood collection tube with DNA/RNA stabilizing reagent</td><td>Zymo Research</td><td>R1150</td><td>DNA/RNA Shield - Blood Collection Tube</td></tr><tr><td>Blunt/TA ligase master mix</td><td>New England Biolabs</td><td>M0367S</td><td>Blunt/TA Ligase Master Mix</td></tr><tr><td>DNA-low binding reaction tube</td><td>Eppendorf</td><td>30108051</td><td>DNA LoBind Tube</td></tr><tr><td>DNase buffer and DNase</td><td>ThermoFisher Scientific</td><td>11766050</td><td>SuperScript&amp;trade; IV VILO&amp;trade; Master Mix with ezDNase&amp;trade; Enzyme</td></tr><tr><td>Flow cell</td><td>Oxford Nanopore Technologies</td><td>FLO-MIN105.24</td><td>flow cell R9.4</td></tr><tr><td>Hot start high fidelity DNA polymerase</td><td>New England Biolabs</td><td>M0493L</td><td>Q5 Hot Start High-Fidelity DNA Polymerase (500 U)</td></tr><tr><td>Magnetic beads</td><td>Beckman Coulter</td><td>A63881</td><td>Agencourt AMPure XP beads</td></tr><tr><td>Magnetic rack</td><td>ThermoFisher Scientific</td><td>12321D</td><td>DynaMag-2 Magnet</td></tr><tr><td>Nanopore sequencing device</td><td>Oxford Nanopore Technologies</td><td>-</td><td>MinION Mk 1B</td></tr><tr><td>PCR barcoding kit</td><td>Oxford Nanopore Technologies</td><td>EXP-PBC001</td><td>PCR Barcoding Kit I (R9)</td></tr><tr><td>Reverse transcriptase master mix</td><td>ThermoFisher Scientific</td><td>11766050</td><td>SuperScript&amp;trade; IV VILO&amp;trade; Master Mix with ezDNase&amp;trade; Enzyme</td></tr><tr><td>RNA purification spin column, DNA/RNA prep buffer, DNA/RNA wash buffer, DNase I, DNA digestion buffer</td><td>Zymo Research</td><td>R1151</td><td>Quick-DNA/RNA Blood Tube Kit</td></tr><tr><td>Rotating mixer</td><td>ThermoFisher Scientific</td><td>15920D</td><td>HulaMixer Sample Mixer</td></tr><tr><td>Taq DNA polymerase</td><td>New England Biolabs</td><td>M0287S</td><td>LongAmp Taq 2x Master Mix</td></tr><tr><td>Ultra II End-prep kit</td><td>New England Biolabs</td><td>E7546S</td><td>NEBNext Ultra II End-Repair/dA-tailing Modul</td></tr><tr><td>UV spectrophotometer</td><td>Implen</td><td>-</td><td>NanoPhotometer</td></tr><tr><td>Vacuum manifold</td><td>Zymo Research</td><td>S7000</td><td>EZ-Vac Vacuum Manifold</td></tr></tbody></table>

sequencing of mrna from whole blood using nanopore sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

In a representative experiment to test the presented protocol we extracted the RNA from 10 different blood samples of five animal species (i.e., 2 individuals per species (goat, sheep, swine, dog, cattle)) (Table 3). RNA yields and quality following extraction can vary widely, in particular due to differences in sample handling and storage. In our representative experiment, we observed RNA concentrations between 43 ng and 543 ng per &#181;L (Table 3). Also, after amplification by RT-PCR, gel analysis of the NPC-1 PCR-products showed various outcomes (Figure 2), with markedly weaker bands for samples BC01 and BC02 (both goat). These differences were most likely caused by differences in sample quality, although differences in PCR efficacy due to differences in primer binding to the NPC1 gene of different species cannot be excluded. However, these differences in yield and/or amplification efficiency did not markedly impact the overall sequencing outcome. Further, an additional non-specific PCR product occurred in sample BC10 (cattle). In contrast to Sanger sequencing, such non-specific products do not negatively influence the results of nanopore sequencing, as these reads are discarded during mapping of the obtained reads to a reference sequence as part of the data analysis.
Prior to each sequencing run, a quality check of the flow cell to be used is strongly recommended, with a minimum requirement of 800 total pores. In our representative experiment, this quality check returned 1,102 pores available for sequencing. Since the data are provided in real time and can be analyzed immediately, the length of a sequencing run can be adjusted for the individual application (i.e., until sufficient sequencing data is produced for the desired analysis). In our experiments, sequencing runs are typically performed overnight, and in the case of our representative experiment we obtained approximately 1.4 million reads during such a 14 h run.
Depending on the type of data analysis to be performed, it can be advisable to process only a subset of the obtained reads. In the case of our representative experiment, a subset of 10,000 reads was selected for further analysis. To this end, the fastq files generated during the sequencing run were further processed in an Ubuntu 18.04 LTS environment, and demultiplexed using flexbar v3.0.3 with parameters optimized for demultiplexing of nanopore sequencing data (barcode-tail-length 300, barcode-error-rate 0.2, barcode-gap-penalty -1)12. After demultiplexing, read mapping and consensus generation can then be done using a number of different tools, but a detailed discussion of the bioinformatics aspect of nanopore sequencing goes beyond the scope of this manuscript. However, in the case of our representative results, read mapping to a reference sequence was performed using Geneious 10.2.3. Of the 10,000 reads analyzed, 5,457 showed a length between 1,750 and 2,000 nucleotides, which matches the expected sizes for the PCR fragments amplified as part of our workflow (1,769 nt, Figure 3). An additional peak in the length distribution of reads was observed between 250 to 500 nucleotides, which can be attributed to unspecific PCR products. Demultiplexing of reads allowed the assignment of 87.6% of the reads to one of the 10 barcodes/samples analyzed (Figure 4). The proportion of demultiplexed reads for each barcode ranged from 3.4% for barcode 1 to 16.9% for barcode 10; however, due to the overall large number of reads this still allowed meaningful consensus calling with a high read depth even for these lower abundance barcode datasets. Indeed, mapping of the sorted reads to a reference sequence of NPC1 resulted in between 31.7% (barcode 2) and 100% (barcode 7 and 8) of reads mapping to the reference, giving a read depth of more than 90 reads at any position for each sample. This is then more than adequate to allow confident consensus base-calling with a negligible error rate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59377/59377fig1.jpg" /> 
Figure 1: Schematic representation of DNA sequencing using nanopore technology. A single-stranded DNA molecule passes through a nanopore embedded in an electrically resistant membrane, with a helicase regulating the transition speed. An ionic current simultaneously passes through the pore and is continuously measured. Modulations of the current caused by the nucleotides present in the pore are detected and computationally back-translated into the nucleotide sequence of the DNA strand. <a href="https://www.jove.com/files/ftp_upload/59377/59377fig1large.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/59377/59377fig2.jpg" /> 
Figure 2: Amplification of PCR products of Niemann-Pick C1 from mRNA. mRNA was isolated from goat (BC01 and 02), sheep (BC03 and 04), swine (BC05 and 06), dog (BC07 and 08), and cattle (BC09 and 10). Nested PCR products were separated in a 0.8% agarose gel in 1x TAE buffer (prepared from 50x TAE buffer: 242.28 g of Tris base, 57.1 mL of glacial acetic acid, 100 mL of 0.5 M EDTA, dH2O to 1 L, pH adjusted to 8.0) for 45 min at 100 V and stained with Sybr Safe. <a href="https://www.jove.com/files/ftp_upload/59377/59377fig2large.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/59377/59377fig3.jpg" /> 
Figure 3: Read-length distribution of 10,000 reads from the representative experiment. The number of reads obtained having a given read length interval is indicated. <a href="https://www.jove.com/files/ftp_upload/59377/59377fig3large.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/59377/59377fig4.jpg" /> 
Figure 4: Distribution of reads after demultiplexing. The number and percentage of demultiplexed (grey) and mapped reads (black) for each barcode are shown. <a href="https://www.jove.com/files/ftp_upload/59377/59377fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Overview of primer sets used. Initial amplification of target sequences was performed with Primer Set 1. Primer Set 2 was then used for nested amplification and adapter addition. Adapters are indicated in red. <a href="https://www.jove.com/files/ftp_upload/59377/table_1.xlsx">Please click here to download this file.</a>
Table 2: Overview of barcode sequences. Individual barcodes were used to identify each sequenced sample. <a href="https://www.jove.com/files/ftp_upload/59377/table_2.xlsx">Please click here to download this file.</a>
Table 3: RNA concentrations obtained following extraction from blood samples sequenced in the representative experiment. The RNA concentrations of two individuals from each of five species are shown, and the ratios of the optical densities at 260/280 nm and 260/230 nm are indicated. <a href="https://www.jove.com/files/ftp_upload/59377/table_3.xlsx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Sequencing of mRNA from Whole Blood using Nanopore Sequencing at JoVE.com

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

sequencing-of-mrna-from-whole-blood-using-nanopore-sequencing

Research

JoVE Journal

Genetics

13.5K Views.  Friedrich-Loeffler-Institut. Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Video: Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Environment

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

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.

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 ...

Bioengineering

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Biology

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Immunology and Infection

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Creating Robust Bisulfite-mRNA Libraries for Transcriptome-Wide Mapping

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.

Author Spotlight: Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study 

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA ...

A Nanopore-Based Genomic Approach to Control Rabies in Low-Income Countries

Whole Genome Sequencing for Rapid Characterization of Rabies Virus Using Nanopore Technology

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance remains limited in many low- and middle-income countries (LMICs), where dog-mediated rabies and/or rabies transmitted by wildlife such as vampire bats pose major public health and economic concerns. We present here a rapid and affordable sample-to-sequence-to-interpretation workflow using nanopore technology. Protocols for sample collection and the diagnosis of rabies are briefly described, followed by details of the optimized whole genome sequencing workflow, including primer design and optimization for multiplex polymerase chain reaction (PCR), a modified, low-cost sequencing library preparation, sequencing with live and offline base calling, genetic lineage designation, and phylogenetic analysis. Implementation of the workflow is demonstrated, and critical steps are highlighted for local deployment, such as pipeline validation, primer optimization, inclusion of negative controls, and the use of publicly available data and genomic tools (GLUE, MADDOG) for classification and placement within regional and global phylogenies. The turnaround time for the workflow is 2-3 days, and the cost ranges from $25 per sample for a 96 sample run to $80 per sample for a 12 sample run. We conclude that setting up rabies virus genomic surveillance in LMICs is feasible and can support progress toward the global goal of zero dog-mediated human rabies deaths by 2030, as well as enhanced monitoring of wildlife rabies spread. Moreover, the platform can be adapted for other pathogens, helping to build a versatile genomic capacity that contributes to epidemic and pandemic preparedness.

Author Spotlight: A Cost-Effective Genomic Workflow for Advancing Rabies Control in Resource-Limited Settings 

Here, we present a rapid and cost-effective workflow for characterizing rabies virus (RABV) genomes using nanopore technology. The workflow is intended to support genomics-informed surveillance at a local level, providing information on circulating RABV lineages and their placement within regional phylogenies to guide rabies control measures.

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic ...

Transcriptome Profiling for High-Throughput Drug Screening

Cost-Efficient Transcriptomic-Based Drug Screening

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

Author Spotlight: Cost-Effective Transcriptomic Drug Screening - Unlocking New Targets 

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease ...