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Sequencing of Ebola Virus Genomes Using Nanopore Technology
采用纳米孔技术对埃博拉病毒测序   

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Abstract

Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation sequencing methods, and was successfully used under outbreak conditions (Hoenen et al., 2016; Quick et al., 2016). Here we describe a protocol used for sequencing of Ebola virus under outbreak conditions using Nanopore technology, which we successfully implemented at the CDC/NIH diagnostic laboratory (de Wit et al., 2016) located at the ELWA-3 Ebola virus Treatment Unit in Monrovia, Liberia, during the recent Ebola virus outbreak in West Africa.

Background

Determining the full-length sequence of virus genomes is an essential procedure in virology. While the classical approach to this involved Sanger sequencing following a primer-walking strategy, newer approaches involve the use of next-generation sequencing methods such as 454, Illumina or Ion Torrent technologies. A common problem with all these technologies, despite their many advantages, is that the required instrumentation is large, expensive, fragile, and therefore difficult to transport. Also, library preparation procedures are often involved. While under usual circumstances these issues are of little consequence, as these machines are run in specialized laboratories with excellent infrastructure, during virus outbreaks in remote areas (for example in case of ebolavirus outbreaks) this can pose significant problems, particularly since the export of samples from affected areas to these specialized laboratories is often politically and logistically challenging. Under these circumstances, the availability of a sequencing technology that can be easily and quickly deployed into remote areas, and allows sequencing to be done directly in an outbreak area, can be invaluable. Therefore, we have tested the MinION sequencing device, which at that time was under development by Oxford Nanopore Technologies (ONT), at the field diagnostic laboratory at the ELWA-3 Ebola virus Treatment Unit in Monrovia (de Wit et al., 2016) during the recent Ebola virus outbreak in West Africa, and developed a protocol for the rapid generation of full-length sequences of Ebola viruses under these conditions. This device employs nanopores, through which nucleotide-strands are transported in a controlled fashion. The nucleotides block and thus modulate an ion-current flowing through those pores, depending on the physical properties of the nucleotides passing through the nanopores, and these current modulations are measured by the device and translated into nucleotide sequences. Results of this test, which indicated that this technology indeed shows great promise as a rapidly deployable and highly usable sequencing platform, are available elsewhere (Hoenen et al., 2016), as are the results of a similar test using the same sequencing platform performed independently of our own efforts by Quick et al. (2016).

Materials and Reagents

Note: This protocol was established and tested during the Ebola virus outbreak in West Africa in January 2015, using materials and reagents available at that time. As the development of the MinION platform progresses rapidly, some modifications might be necessary to adopt the protocol to the materials and reagents available now. Particularly, while at the time the technology was only available to members of the MinION access program, it is now commercially available.

  1. 0.2 ml PCR-tubes (ideally in strips of 8) (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: AB0490 )
  2. 1.5 ml tubes (e.g., Thermo Fisher Scientific, Fisher Scientific, catalog number: S348903 )
  3. Eppendorf protein LoBind tubes, 1.5 ml, PCR clean (Eppendorf, catalog number: 0030108116 )
  4. Gloves
  5. MinION flowcell, revision 7.3 (Oxford Nanopore Technologies [ONT])
  6. RNA freshly purified (or stored at -80 °C) from patient blood samples following appropriate safety protocols
  7. SuperScript III First-Strand-Synthesis System (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080051 )
  8. DEPC-treated nuclease-free water (e.g., Thermo Fisher Scientific, AmbionTM, catalog number: AM9906 )
  9. Primer, 10 μM (see Table 1 for sequences); primer CGGACACACAAAAAGAAAGAAG at a concentration of 2 μM and 10 μM
  10. dNTP mix, 10 mM each (e.g., New England Biolabs, catalog number: N0447S )
  11. iProofTM high-fidelity polymerase (Bio-Rad Laboratories, catalog number: 1725301 )
  12. Agencourt AMPure XP beads (Beckman Coulter, catalog number: A63881 )
  13. His-Tag dynabeads (Thermo Fisher Scientific, NovexTM, catalog number: 10103D )
  14. Magnetic stand for PCR-purification with Agencourt AMPure XP beads in 1.5 ml tubes (e.g., Beckman Coulter, catalog number: A29182 )
  15. 70% ethanol
  16. Qiagen elution buffer (QIAGEN, catalog number: 19086 )
  17. 50x TAE buffer (ideally in 10.2 ml aliquots) (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: B49 )*
  18. Agarose (ideally in pre-weighed aliquots of 0.5 g) (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500100 )*
  19. DNA standard (e.g., New England Biolabs, catalog number: N3200S )*
  20. 6x gel loading dye (e.g., New England Biolabs, catalog number: B7022S )*
  21. Fast Blast DNA stain (Bio-Rad Laboratories, catalog number: 1660420 )*
  22. NEBNext dA-tailing module (New England Biolabs, catalog number: E6053S )
  23. NEBNext end repair module (New England Biolabs, catalog number: E6050S )
  24. Blunt/TA ligase master mix (New England Biolabs, catalog number: M0367L )
  25. Genomic DNA Sequencing Kit SQK-MAP004 (ONT): contains DNA CS, 2x wash buffer, elution buffer, EP buffer, HP adapter, Adapter mix and Fuel mix

*Note: Optional materials and reagents.

Equipment

  1. MinION sequencing device (ONT)
  2. PCR cycler (e.g., Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: A24811 )
  3. Vortexer (e.g., Thermo Fisher Scientific, Fisher Scientific, catalog number: S96461A )
  4. Mini centrifuges for 1.5 and 0.2 ml tubes (e.g., VWR, catalog number: 93000-204 )
  5. Pipettes: 10 μl, 20 μl, 100 μl, 1,000 μl, 8 x 20 μl multichannel with filter tips (e.g., Mettler-Toledo, catalog numbers: PR-10 , PR-20 , PR-100 , PR-1000 , L8-20XLS+ )
  6. Racks for 0.2 ml PCR tubes and 1.5 ml tubes (e.g., Thermo Fisher Scientific, Fisher Scientific, catalog numbers: 05-541-85 , 05-541-2 )
  7. PCR cooler rack, 0.2 ml, 4 °C (e.g., Eppendorf, catalog number: 3881000031 )
  8. Styrofoam box with lid and cold packs (frozen cold pack at bottom, 4 °C cold pack on top, then sample rack on top of that) as cooler box (or alternatively ice box, when ice is available)
  9. Electrophoresis chamber with power supply (e.g., Mupid-exU, Takara Clontech)*
  10. Microwave oven*
  11. Agarose gel casting stand, or alternatively laboratory tape*
  12. 250-300 ml glass flask for preparing agarose*
  13. Box for agarose gel staining*
  14. Additional infrastructure when sequencing under outbreak conditions
    Note: This infrastructure is a given in any Western laboratory, but should be considered when establishing the method under field conditions in outbreak areas. While this topic cannot be exhaustively covered in this protocol, the following points should be carefully considered.
    1. Power generator, uninterruptable power supplies, voltage regulators (all depending on the quality of the electricity supply).
    2. Air conditioning if possible, alternatively external heatsink (e.g., a ~30 x 30 cm metal plate) for MinION sequencing device.
    3. Clean water for preparation of 1x TAE, and for DNA staining and destaining - bottled drinking water (ideally in 500 ml bottles) can be used for this purpose if no other source of clean water is available.
    4. Equipment for safe sample inactivation and RNA extraction.
    5. 4 °C fridge, -20 °C freezer, optionally -80 °C freezer for storage of RNA.
    6. Personal protective equipment as deemed necessary.
    *Note: Optional equipment.

Software

  1. Laptop running MinKNOW software v 0.48.2.12 (ONT) with internet connection (e.g., via 3G wireless router); for current IT requirements please check the ONT internet site (https://nanoporetech.com/community/faqs).

Procedure

  1. Reverse transcription (RT) using the SuperScript III First-Strand-Synthesis System
    1. Combine 4 μl RNase free water, 1 μl primer (2 μM, CGGACACACAAAAAGAAAGAAG), 1 μl dNTPs, 5 μl RNA, mix by flicking, spin down, incubate for 5 min at 65 °C, put in PCR cooler for > 1 min.
    2. Prepare mastermix (6 μl 5x FS buffer, 1.5 μl DTT, 1.5 μl RNase out, 1.5 μl Superscript III. All components are provided as part of the RT kit), add 7 μl to samples, mix by flicking, spin down, incubate for 50 min at 50 °C , 5 min at 85 °C, hold at 4 °C, add 1 μl RNase H, mix by flicking, spin down, incubate for 20 min at 37 °C, hold at 4 °C.

  2. PCR using iProof polymerase
    1. Set up mastermix for 14 reactions (Table 2; this includes 2 extra reactions that are not used) using the cDNA from step A2 as template, dispense 12 x 44.2 μl into 0.2 ml PCR tube strips on PCR cooler block, add 2.5 μl forward primer (10 μM) and 2.5 μl reverse primer (10 μM) according to table 1, flick, spin down, cycle (Table 3, first PCRs).
    2. Set up mastermix for 28 reactions (Table 2; this includes 4 extra reactions that are not used) using the PCR products from step B1 as template, dispense 24 x 44.2 μl into PCR tube strips on PCR cooler block, add 0.8 μl PCR product from step B1 as template (see Table 1 to determine which PCR product from step B1 to use as template), add 2.5 μl forward primer (10 μM) and 2.5 μl reverse primer (10 μM) according to Table 1, flick, spin down, cycle (Table 3, second PCRs). For an overview of the whole procedure see Figure 1.

      Table 1. Primer sequences. Each pair of primer sequences (1 forward and 1 reverse primer) correspond to a single reaction.


      Table 2. PCR master mix


      Table 3. PCR cycling conditions



      Figure 1. Schematic of PCR amplification prior to library preparation

  3. Agencourt PCR-purification
    1. Vortex AMPure XP beads for 10 sec.
    2. Out of the 24 PCR products, generate two pools of PCR products (Figure 1; the first pool contains PCR products 1.1-6.2, and the second pool PCR products 7.1-12.2), each containing 12 x 40 μl, for a total of 480 μl each, and pipet into 1.5 ml Eppendorf tube.
    3. Add 720 μl beads to the pools, mix by pipetting 10 times, incubate for 5 min.
    4. Place tube onto magnet, wait 5 min.
    5. Take off supernatant completely, being careful not to aspirate any beads, add 1,200 μl 70% EtOH (do NOT pipet directly onto beads), let sit for at least 30 sec, take off completely with a P1000 pipette.
    6. Repeat wash step with 800 μl 70% EtOH, take off supernatant with a P1000 pipette, spin down for 3 sec, put back on magnet, take off any remaining supernatant with a P10 pipette.
    7. Dry beads for about 2 min with open lids, keep lids open from now on.
    8. Take tube off magnet, add 60 μl Qiagen elution buffer, resuspend bead-pellet in elution buffer, incubate for 10 min at room temperature.
    9. Place back onto magnet, wait 1 min (until beads have pelleted), take off 50 μl of eluate. Pool the eluates from the two purified PCR product pools, for a total of 100 μl containing all PCR products.

  4. Optional: Quality control on agarose gel
    1. Prepare 1x TAE by adding 10.2 ml 50x TAE into 500 ml water.
    2. Combine 50 ml 1x TAE and 0.5 g agarose, bring to boil in microwave.
    3. Pour agarose gel, let solidify on a cold pack.
    4. Load 15 μl of DNA ladder and 5 μl of pooled sample mixed with 1 μl 6x loading dye onto agarose gel, run for about 20 min at 100 V.
    5. Combine 50 ml FastBlast DNA stain and 200 ml water, incubate the agarose gel for 3 min in the diluted DNA stain, then rinse and destain the gel with several changes of water, rocking the gel continuously for 10 to 20 min.
    6. Visualize the gel using a white laptop screen as lightbox. Several discrete bands should be visible, corresponding to the various PCR product lengths (strong band at ~2.1 kb, moderate band at ~2.7 kb, weak bands at 1.3, 3.4, and 4.0 kb).

  5. Library preparation
    1. Combine 80 μl of pooled, purified PCR products from step C9 and 5 μl DNA CS, add 10 μl 10x NEBNext end repair buffer and 5 μl NEBNext end repair enzyme mix, mix by pipetting, incubate at RT for 30 min.
    2. Clean up with 180 μl Agencourt beads as described in section C, but use 300 μl EtOH for washing, add 30 μl elution buffer, take off 25 μl eluate, put in PCR tube.
    3. Add 3 μl 10x NEBNext dA-tailing buffer and 2 μl NEBNext dA-tailing enzyme mix, incubate for 30 min at 37 °C.
    4. Thaw 2x wash buffer, elution buffer, EP buffer at RT, thaw HP adapter, Adapter mix and Fuel mix in the cooler box.
    5. Prepare His-beads:
      1. Mix 550 μl 2x wash buffer and 550 μl nuclease-free water by inverting 10x, briefly spin down.
      2. Vortex His-Beads for ~30 sec.
      3. Pipet 10 μl His-Beads into a LoBind tube, add 250 μl 1x wash buffer.
      4. Place on magnet, wait for pellet to form, aspirate off supernatant, add 250 μl wash buffer, wait 30 sec, aspirate off supernatant.
      5. Take tube off magnet, resuspend pellet in 100 μl 2x wash buffer.
    6. After 30 min, in a LoBind tube, add (directly to the bottom of the tube) 8 μl nuclease-free water, 30 μl DNA from step E3, 10 μl Adapter mix, 2 μl HP Adapter, and 50 μl Blunt/TA ligase master mix, mix by pipetting, if necessary briefly spin down, incubate 10 min at RT.
    7. Add 100 μl washed beads (directly to bottom of tube), carefully mix by pipetting, incubate for 5 min at RT.
    8. Place on magnet, wait 2 min for pellet to form, aspirate supernatant, wash 2 x for 30 sec each with 250 μl 1x wash buffer.
    9. With the lid close, briefly centrifuge, put back onto magnet, wait 1 min with lid closed, aspirate off any remaining wash buffer.
    10. Take off magnet, resuspend pellet in 25 μl elution buffer (adding the elution buffer directly to the pellet), wait 10 min with lid closed, put back on magnet, wait 1 min, take off 15 μl eluate (≥ pre-sequencing mix).

  6. Loading of library (Figure 2)
    1. Mix 318.5 μl EP buffer and 6.5 μl Fuel mix by vortexing, spin down.
    2. Prime flow cell two times with 150 μl of EB/Fuel mix.
    3. Mix 6 μl library, 141 μl EP, 3 μl Fuel mix by inverting 10 times, briefly spin down, load, and initiate flow cell run.


      Figure 2. Priming/loading of the flowcell

Data analysis

Once the sequencing run is completed on the MinION device, raw data have to be base-called, primer sequences have to be removed, and then all sequences have to be aligned to a reference sequence to build a pile-up, which can then be used for consensus-calling. The exact procedure for doing so, including all used bioinformatics scripts, is published in (Hoenen et al., 2016), which is openly accessible at this link: http://wwwnc.cdc.gov/eid/article/22/2/15-1796_article.

Acknowledgments

The author is grateful to all members of the former NIH/CDC diagnostic laboratory at ELWA3, Monrovia, Liberia. This work was funded in part by the Intramural Research Program of the National Institutes of Health, NIAID. The author was participant of the MinION access program, and received some of the flowcells and reagents used from ONT free of charge or at reduced cost. The author was invited by ONT to present part of this work at the ‘London Calling’ 2015 meeting organized by ONT in London, U.K.

References

  1. de Wit, E., Falzarano, D., Onyango, C., Rosenke, K., Marzi, A., Ochieng, M., Juma, B., Fischer, R. J., Prescott, J. B., Safronetz, D., Omballa, V., Owuor, C., Hoenen, T., Groseth, A., van Doremalen, N., Zemtsova, G., Self, J., Bushmaker, T., McNally, K., Rowe, T., Emery, S. L., Feldmann, F., Williamson, B., Nyenswah, T. G., Grolla, A., Strong, J. E., Kobinger, G., Stroeher, U., Rayfield, M., Bolay, F. K., Zoon, K. C., Stassijns, J., Tampellini, L., de Smet, M., Nichol, S. T., Fields, B., Sprecher, A., Feldmann, H., Massaquoi, M. and Munster, V. J. (2016). The merits of malaria diagnostics during an Ebola virus disease outbreak. Emerg Infect Dis 22(2): 323-326.
  2. Hoenen, T., Groseth, A., Rosenke, K., Fischer, R. J., Hoenen, A., Judson, S. D., Martellaro, C., Falzarano, D., Marzi, A., Squires, R. B., Wollenberg, K. R., de Wit, E., Prescott, J., Safronetz, D., van Doremalen, N., Bushmaker, T., Feldmann, F., McNally, K., Bolay, F. K., Fields, B., Sealy, T., Rayfield, M., Nichol, S. T., Zoon, K. C., Massaquoi, M., Munster, V. J. and Feldmann, H. (2016). Nanopore sequencing as a rapidly deployable Ebola outbreak tool. Emerg Infect Dis 22(2): 331-334.
  3. Quick, J., Loman, N. J., Duraffour, S., Simpson, J. T., Severi, E., Cowley, L., Bore, J. A., Koundouno, R., Dudas, G., Mikhail, A., Ouedraogo, N., Afrough, B., Bah, A., Baum, J. H., Becker-Ziaja, B., Boettcher, J. P., Cabeza-Cabrerizo, M., Camino-Sanchez, A., Carter, L. L., Doerrbecker, J., Enkirch, T., Garcia-Dorival, I., Hetzelt, N., Hinzmann, J., Holm, T., Kafetzopoulou, L. E., Koropogui, M., Kosgey, A., Kuisma, E., Logue, C. H., Mazzarelli, A., Meisel, S., Mertens, M., Michel, J., Ngabo, D., Nitzsche, K., Pallasch, E., Patrono, L. V., Portmann, J., Repits, J. G., Rickett, N. Y., Sachse, A., Singethan, K., Vitoriano, I., Yemanaberhan, R. L., Zekeng, E. G., Racine, T., Bello, A., Sall, A. A., Faye, O., Faye, O., Magassouba, N., Williams, C. V., Amburgey, V., Winona, L., Davis, E., Gerlach, J., Washington, F., Monteil, V., Jourdain, M., Bererd, M., Camara, A., Somlare, H., Camara, A., Gerard, M., Bado, G., Baillet, B., Delaune, D., Nebie, K. Y., Diarra, A., Savane, Y., Pallawo, R. B., Gutierrez, G. J., Milhano, N., Roger, I., Williams, C. J., Yattara, F., Lewandowski, K., Taylor, J., Rachwal, P., Turner, D. J., Pollakis, G., Hiscox, J. A., Matthews, D. A., O'Shea, M. K., Johnston, A. M., Wilson, D., Hutley, E., Smit, E., Di Caro, A., Wolfel, R., Stoecker, K., Fleischmann, E., Gabriel, M., Weller, S. A., Koivogui, L., Diallo, B., Keita, S., Rambaut, A., Formenty, P., Gunther, S. and Carroll, M. W. (2016). Real-time, portable genome sequencing for Ebola surveillance. Nature 530(7589): 228-232.

简介

病毒基因组在疾病爆发期间的测序可以为诊断,流行病学和潜在对策的评估提供有价值的信息。然而,特别是在偏远地区,后勤和技术挑战可能很大。纳米孔测序提供了经典的Sanger和下一代测序方法的替代物,并且在爆发条件下成功使用(Hoenen等人,2016; Quick等人,2016 )。在这里我们描述了用于在爆发条件下使用纳米孔技术对埃博拉病毒进行测序的方案,我们在CDC/NIH诊断实验室(de Wit等人,2016)成功地实施了ELWA- 3 确定病毒基因组的全长序列是必不可少的,因为它是在利比里亚蒙罗维亚的埃博拉病毒治疗股在最近在西非爆发的埃博拉病毒爆发期间。

<病毒学中的程序。虽然经典的方法涉及Sanger测序后引物步行战略,较新的方法涉及使用下一代测序方法,如454,Illumina或Ion Torrent技术。所有这些技术的常见问题,尽管它们的许多优点,是所需的仪器是大的,昂贵的,脆弱的,因此难以运输。此外,经常涉及文库制备程序。虽然在通常情况下这些问题没有什么影响,因为这些机器在具有优良基础设施的专门实验室中运行,在偏远地区的病毒爆发期间(例如在埃博拉病毒爆发的情况下),这可能造成重大问题,特别是在样品出口从受影响的地区到这些专门的实验室往往在政治和后勤方面具有挑战性。在这些情况下,可以容易和快速地部署到偏远地区,并允许在爆发区域直接进行测序的测序技术的可用性是非常有价值的。因此,我们已经在Monrovia的ELWA-3埃博拉病毒治疗单元的现场诊断实验室测试了MinION测序装置,该装置当时正在由Oxford Nanopore Technologies(ONT)开发。(de Wit等人,/em>。,2016),并且制定了在这些条件下快速产生埃博拉病毒的全长序列的方案。该装置使用纳米孔,核苷酸链通过其以受控的方式运输。核苷酸阻断并因此调节流过这些孔的离子流,这取决于通过纳米孔的核苷酸的物理性质,并且这些电流调节通过装置测量并翻译成核苷酸序列。该测试的结果表明该技术确实显示出作为可快速部署和高度可用的测序平台的巨大前景,其在其他地方是可用的(Hoenen等人,2016),以及类似的使用与Quick自己的努力独立地执行的相同测序平台进行的测试。 (2016年)。

材料和试剂

注意:该协议是在2015年1月在西非埃博拉病毒爆发期间使用当时可用的材料和试剂建立和测试的。随着Minion平台的发展迅速,可能需要进行一些修改,以便采用目前可用的材料和试剂的协议。特别地,虽然当时该技术仅提供给MinION接入程序的成员,但现在已经可以商业化。

  1. 0.2ml PCR管(理想地为8条)(例如,Thermo Fisher Scientific,Thermo Scientific TM ,目录号:AB0490)。
  2. 1.5ml管(例如,Thermo Fisher Scientific,Fisher Scientific,目录号:S348903)
  3. Eppendorf蛋白LoBind管,1.5ml,PCR清洗(Eppendorf,目录号:0030108116)
  4. 手套
  5. Minion flowcell,revision 7.3(Oxford Nanopore Technolgies [ONT])
  6. 根据适当的安全规程从患者血液样品新鲜纯化(或储存于-80℃)的RNA
  7. SuperScript III第一链合成系统(Thermo Fisher Scientific,Invitrogen TM ,目录号:18080051)
  8. DEPC处理的无核酸酶的水(例如Thermo Fisher Scientific,Ambion TM ,目录号:AM9906)
  9. 引物,10μM(序列见表1);引物CGGACACACAAAAAGAAAGAAG,浓度为2μM和10μM
  10. dNTP混合物,各10mM(例如,New England Biolabs,目录号:N0447S)
  11. iProof TM sup/high高保真聚合酶(Bio-Rad Laboratories,目录号:1725301)
  12. Agencourt AMPure XP珠(Beckman Coulter,目录号:A63881)
  13. His-Tag dynabeads(Thermo Fisher Scientific,Novex TM ,目录号:10103D)
  14. 用于在1.5ml管(例如,Beckman Coulter,目录号:A29182)中用Agencourt AMPure XP珠粒进行PCR纯化的磁性架子
  15. 70%乙醇
  16. Qiagen洗脱缓冲液(QIAGEN,目录号:19086)
  17. 50x TAE缓冲液(理想地为10.2ml等分试样)(例如Thermo Fisher Scientific,Thermo Scientific TM ,目录号:B49)*
  18. 琼脂糖(理想地,在0.5g的预称重等分试样中)(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:16500100)*
  19. DNA标准(例如,New England Biolabs,目录号:N3200S)*
  20. 6x凝胶负载染料(例如,New England Biolabs,目录号:B7022S)*
  21. 快速blast DNA染色(Bio-Rad Laboratories,目录号:1660420)*
  22. NEBNext dA-tailing module(New England Biolabs,目录号:E6053S)
  23. NEBNext末端修复模块(New England Biolabs,目录号:E6050S)
  24. Blunt/TA连接酶主混合物(New England Biolabs,目录号:M0367L)
  25. 基因组DNA测序试剂盒SQK-MAP004(ONT):包含DNA CS,2x洗涤缓冲液,洗脱缓冲液,EP缓冲液,HP适配器,接头混合物和燃料混合物

*注:可选材料和试剂。

设备

  1. MinION定序装置(ONT)
  2. PCR循环仪(例如Thermo Fisher Scientific,Applied Biosystems TM ,目录号:A24811)。
  3. Vortexer(例如,Thermo Fisher Scientific,Fisher Scientific,目录号:S96461A)。
  4. 用于1.5和0.2ml管(例如,VWR,目录号:93000-204)的微型离心机
  5. 移液管:10μl,20μl,100μl,1,000μl,8×20μl具有过滤嘴的多通道(例如,Mettler-Toledo,目录号:PR-10,PR-20, 100,PR-1000,L8-20XLS +)
  6. 用于0.2ml PCR管和1.5ml管(例如,Thermo Fisher Scientific,Fisher Scientific,目录号:05-541-85,05-541-2)的架子
  7. PCR冷却器架,0.2ml,4℃(例如,Eppendorf,目录号:3881000031)。
  8. 带有盖子和冷包装的聚苯乙烯泡沫塑料盒(底部为冷冻冷包,顶部为4°C冷包装,然后顶部为样品架)作为冷却器盒(或冰块可用时为冰盒)
  9. 具有电源的电泳室(例如,Mupid-exU,Takara Clontech)*
  10. 微波炉*
  11. 琼脂糖凝胶铸造台,或替代实验室胶带*
  12. 250-300ml用于制备琼脂糖的玻璃烧瓶*
  13. 用于琼脂糖凝胶染色的盒*
  14. 在爆发情况下排序时的其他基础设施
    注意:这种基础设施是在任何西方实验室给定的,但是在暴发地区在现场条件下建立方法时应考虑。虽然本主题不能在本协议中详尽涵盖,但以下几点应该仔细考虑。
    1. 发电机,不间断电源,稳压器(都取决于电源的质量)。
    2. 空调(如果可能),或者外部散热器(例如,?30 x 30厘米的金属板),用于MinION顺序装置。
    3. 用于制备1x TAE的清水,以及用于DNA染色和脱色的瓶装饮用水(理想情况下为500ml瓶),如果没有其他来源的清水,可用于此目的。
    4. 用于安全样品灭活和RNA提取的设备
    5. 4℃冰箱,-20℃冰箱,任选-80℃冰箱用于储存RNA
    6. 必要时采取个人防护设备。
    *注意:可选设备。

软件

  1. 通过3G无线路由器运行具有互联网连接(例如。)的MinKNOW软件v 0.48.2.12(ONT)的膝上型计算机;对于当前的IT要求,请检查ONT网站( https://nanoporetech.com/community/faqs )。

程序

  1. 使用SuperScript III第一链合成系统的逆转录(RT)
    1. 将4μl无RNA酶的水,1μl引物(2μM,CGGACACACAAAAAGAAAGAAG),1μldNTP,5μlRNA,通过轻敲,旋转混合,在65℃下温育5分钟混合,加入PCR冷却器中, 1分钟。
    2. 准备mastermix(6μl5x FS缓冲液,1.5μlDTT,1.5μlRNAse输出,1.5μlSuperscript III。所有组分均作为RT试剂盒的一部分提供),向样品中加入7μl,通过轻弹,旋转混合,孵育50℃50分钟,85℃5分钟,保持在4℃,加入1μlRNAse H,通过轻敲,旋转混合,在37℃下孵育20分钟,保持在4℃。 />
  2. 使用iProof聚合酶进行PCR
    1. 使用来自步骤A2的cDNA作为模板,对于14个反应(表2;包括2个未使用的额外反应)设置主混合物,将12×44.2μl分配到PCR冷却器块上的0.2ml PCR管条中,加入2.5μl正向引物(10μM)和2.5μl反向引物(10μM),根据表1,轻敲,离心,循环(表3,第一PCR)。
    2. 使用来自步骤B1的PCR产物作为模板设置28个反应(表2;这包括4个未使用的额外反应)的mastermix,将24×44.2μl分配到PCR冷却器块上的PCR管条中,加入0.8μlPCR产物步骤B1作为模板(参见表1,确定来自步骤B1的PCR产物用作模板),根据表1加入2.5μl正向引物(10μM)和2.5μl反向引物(10μM),轻敲,循环(表3,第二PCR)。有关整个过程的概述,请参见图1.

      表1.引物序列。每对引物序列(1个正向引物和1个反向引物)对应一个反应。


      表2. PCR主混合


      表3. PCR循环条件



      图1.文库制备前的PCR扩增示意图

  3. Agencourt PCR纯化
    1. Vortex AMPure XP珠10秒。
    2. 在24个PCR产物中,产生两个PCR产物池(图1;第一池包含PCR产物1.1-6.2,和第二池PCR产物7.1-12.2),每个包含12×40μl,总共480 μl,并移液至1.5ml Eppendorf管中
    3. 向池中加入720μl珠子,通过吸移混合10次,孵育5分钟
    4. 将管放在磁铁上,等待5分钟。
    5. 小心取出上清液,小心不要吸出任何珠子,加入1,200μl70%EtOH(不要直接吸到珠子上),让其静置至少30秒,用P1000移液管完全取出。
    6. 用800μl70%EtOH重复洗涤步骤,用P1000移液管取出上清液,旋转3秒,放回磁铁,用P10移液管取出任何剩余的上清液。
    7. 干珠约2分钟,打开盖子,从现在开始盖子打开
    8. 取管离开磁铁,加入60μlQiagen洗脱缓冲液,将珠粒沉淀重悬在洗脱缓冲液中,在室温下孵育10分钟。
    9. 放回磁铁,等待1分钟(直到珠粒沉淀),取出50μl的洗脱液。汇集来自两个纯化的PCR产物库的洗脱液,总共100μl含有所有PCR产物
  4. 可选:琼脂糖凝胶的质量控制
    1. 通过将10.2 ml 50x TAE加入500 ml水中制备1x TAE
    2. 合并50ml 1x TAE和0.5g琼脂糖,在微波炉中煮沸。
    3. 倒入琼脂糖凝胶,冷冻固化
    4. 加载15μlDNA梯子和5μl混合样品与1μl6x加载染料在琼脂糖凝胶上,在100V运行约20分钟。
    5. 结合50ml FastBlast DNA染色和200ml水,在稀释的DNA染色中孵育琼脂糖凝胶3分钟,然后用几次变化的水冲洗和脱色凝胶,连续摇动凝胶10至20分钟。
    6. 使用白色笔记本电脑屏幕作为lightbox可视化凝胶。应该可以看到几个离散条带,对应于各种PCR产物长度(?2.1kb的强条带,?2.7kb的中等条带,1.3,3.4和4.0kb的弱条带)。

  5. 图书馆准备
    1. 将80μl来自步骤C9的合并的纯化的PCR产物与5μlDNA CS组合,加入10μl10×NEBNext末端修复缓冲液和5μlNEBNext末端修复酶混合物,通过移液混合,在室温下孵育30分钟。
    2. 用180μlAgencourt珠清洗,如C部分所述,但使用300μlEtOH洗涤,加入30μl洗脱缓冲液,取出25μl洗脱液,放入PCR管中。
    3. 加入3微升10x NEBNext dA尾缓冲液和2微升NEBNext dA尾酶混合物,在37℃孵育30分钟。
    4. 解冻2x洗涤缓冲液,洗脱缓冲液,EP缓冲液在RT,解冻HP适配器,适配器混合和燃料混合在冷却器盒。
    5. 准备His珠:
      1. 混合550μl2×洗涤缓冲液和550μl无核酸酶水倒置10x,短暂旋转下来
      2. 漩涡他的珠子?30秒。
      3. 吸取10μlHis-Beads到LoBind管中,加入250μl1x洗涤缓冲液
      4. 放在磁铁上,等待沉淀形成,吸出上清液,加入250μl洗涤缓冲液,等待30秒,吸出上清液。
      5. 取管离开磁铁,将沉淀重悬在100μl2×洗涤缓冲液中
    6. 30分钟后,在LoBind管中,加入(直接到管底部)8μl无核酸酶水,30μl来自步骤E3的DNA,10μl衔接子混合物,2μlHP Adaptor和50μlBlunt/TA连接酶主混合物,通过移液混合,如果必要,短暂离心,在室温下孵育10分钟
    7. 加入100μl洗涤的珠子(直接到管底部),通过吸移小心混合,在室温下孵育5分钟。
    8. 放在磁铁上,等待2分钟以形成沉淀,吸出上清液,用250μl1x洗涤缓冲液洗涤2次,每次30秒。
    9. 将盖子关闭,短暂离心,放回磁铁,等待1分钟,盖子关闭,吸出任何剩余的洗涤缓冲液。
    10. 取出磁铁,在25μl洗脱缓冲液(将洗脱缓冲液直接加入沉淀)中重悬沉淀,等待10分钟,盖上盖子,放回磁铁,等待1分钟,取出15μl洗脱液(≥预测序混合液) 。

  6. 加载库(图2)
    1. 通过涡旋混合318.5μlEP缓冲液和6.5μl燃料混合物,旋转下来
    2. 用150μlEB /燃料混合物冲洗流动池两次。
    3. 混合6微升库,141微升EP,3微升燃料混合通过颠倒10次,短暂下降,加载,并启动流动池运行。


      图2.启动/加载flowcell

数据分析

一旦在Minion设备上完成测序运行,原始数据必须被碱基化,引物序列必须被去除,然后所有序列必须与参考序列比对以构建堆积,然后可以用于共识调用。这样做的确切程序,包括所有使用的生物信息学脚本,公开在(Hoenen等人。,2016),这是公开访问在此链接: http://wwwnc.cdc.gov/eid/article/22/2/15- 1796_article

致谢

作者感谢位于利比里亚蒙罗维亚ELWA3的前NIH/CDC诊断实验室的所有成员。这项工作部分资助的国家卫生研究院,NIAID的校内研究计划。作者参与了Minion接入计划,并免费或以降低的成本接收了ONT使用的一些流通池和试剂。作者受到ONT的邀请,在ONT在伦敦,英国组织的"London Calling"2015年会议上介绍了这部分工作。

参考文献

  1. De Witho,E.,Falzarano,D.,Onyango,C.,Rosenke,K.,Marzi,A.,Ochieng,M.,Juma,B.,Fischer,RJ,Prescott,JB,Safronetz,D.,Omballa V.,Owuor,C.,Hoenen,T.,Groseth,A.,van Doremalen,N.,Zemtsova,G.,Self,J.,Bushmaker,T.,McNally,K.,Rowe, Emery,SL,Feldmann,F.,Williamson,B.,Nyenswah,TG,Grolla,A.,Strong,JE,Kobinger,G.,Stroeher,U.,Rayfield,M.,Bolay,FK,Zoon, Stassijns,J.,Tampellini,L.,de Smet,M.,Nichol,ST,Fields,B.,Sprecher,A.,Feldmann,H.,Massaquoi,M.and Munster,VJ(2016) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26814608"target ="_ blank">在埃博拉病毒病爆发期间疟疾诊断的优点。 Emerg Infect Dis 22(2):323-326。
  2. Hoenen,T.,Groseth,A.,Rosenke,K.,Fischer,RJ,Hoenen,A.,Judson,SD,Martellaro,C.,Falzarano,D.,Marzi,A.,Squires,RB,Wollenberg,KR ,de Wit,E.,Prescott,J.,Safronetz,D.,van Doremalen,N.,Bushmaker,T.,Feldmann,F.,McNally,K.,Bolay,FK,Fields,B.,Sealy,T 。,Rayfield,M.,Nichol,ST,Zoon,KC,Massaquoi,M.,Munster,VJ和Feldmann,H。(2016)。  纳米孔测序作为可快速部署的埃博拉疫情工具 Emerg Infect Dis 22(2):331 -334。
  3. Quick,J.,Loman,NJ,Duraffour,S.,Simpson,JT,Severi,E.,Cowley,L.,Bore,JA,Koundouno,R.,Dudas,G.,Mikhail,A.,Ouedraogo,N B.,Boettcher,JP,Cabeza-Cabrerizo,M.,Camino-Sanchez,A.,Carter,LL,Doerrbecker,J。, Enkirch,T.,Garcia-Dorival,I.,Hetzelt,N.,Hinzmann,J.,Holm,T.,Kafetzopoulou,LE,Koropogui,M.,Kosgey,A.,Kuisma,E.,Logue, Marchens,M.,Michel,J.,Ngabo,D.,Nitzsche,K.,Pallasch,E.,Patrono,LV,Portmann,J.,Repits,JG,Rickett, NY,Sachse,A.,Singethan,K.,Vitoriano,I.,Yemanaberhan,RL,Zekeng,EG,Racine,T.,Bello,A.,Sall,AA,Faye,O.,Faye,O.,Magassouba ,N.,Williams,CV,Amburgey,V.,Winona,L.,Davis,E.,Gerlach,J.,Washington,F.,Monteil,V.,Jourdain,M.,Bererd,M., A.,Somlare,H.,Camara,A.,Gerard,M.,Bado,G.,Baillet,B.,Delaune,D.,Nebie,KY,Diarra,A.,Savane,Y.,Pallawo,RB ,Gutierrez,GJ,Milhano,N.,Roger,I.,Williams,CJ,Yattara,F.,Lewandowski,K.,Taylor,J.,Rachwal,P.,Turner,DJ,Pollakis,G.,Hiscox, JA,Matthews,DA,O'Shea,MK,Johnston,AM,Wilson,D.,Hutley,E.,Smit,E.,Di Caro,A.,Wolfel,R.,Stoecker,K.,Fleischmann,E 。,Gabriel,M.,Weller,SA,Koivogui,L.,Diallo,B.,Keita,S.,Rambaut,A.,Formenty,P.,Gunther,S.and Carroll,MW(2016) 用于埃博拉监测的实时,便携式基因组测序。 530(7589):228-232。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hoenen, T. (2016). Sequencing of Ebola Virus Genomes Using Nanopore Technology. Bio-protocol 6(21): e1998. DOI: 10.21769/BioProtoc.1998.
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