Ebola virus sequencing kit-list
Nanopore | amplicon | native barcoding
| Document: | ARTIC-EBOV-seqKit-v2.0.0 |
| Creation Date: | 2018-05-21 |
| Last Updated: | Sept 25, 2019 |
| Forked from: | doi:10.1038/nprot.2017.066 |
| Author: | Luke Meredith, Josh Quick |
| Licence: | Creative Commons Attribution 4.0 International License |
Overview: The following protocol is adapted from the methods of Quick et al. (2017) Nature Protocols 12: 1261–1276 doi:10.1038/nprot.2017.066 and covers primers, amplicon preparation and clean-up, then uses a single-tube protocol to barcode and adaptor ligate the library, before running minION.
- This document is part of the Ebola virus Nanopore sequencing protocol package:
- http://artic.network/ebov/
Related documents:
- Ebola primer scheme:
- https://github.com/artic-network/primer-schemes/tree/master/ZaireEbola/V2
- Ebola virus Nanopore sequencing protocol:
- http://artic.network/viruses/ebov/ebov-seq-sop.html
- Ebola virus Nanopore sequencing kit-list:
- http://artic.network/ebov/ebov-seq-kit.html
Funded by the Wellcome Trust
Collaborators Award 206298/Z/17/Z --- ARTIC network
Library Preparation and Sequencing Reagents:
| Product | Supplier | Cat No. | Unit Cost | # Req | Cost | |
|---|---|---|---|---|---|---|
| Ebola Zaire V3 Primers | IDT | £450 | 1 | £450 | ||
| SuperScript IV Reverse Transcriptase | Thermo | 18090050 | £230 | 1 | £230 | |
| dNTP mix (10 mM each) | Thermo | R0192 | £82 | 1 | £82 | |
| RNase OUT | Thermo | 10777019 | £163 | 1 | £163 | |
| Q5® Hot Start HF Polymerase (2xMM) | NEB | M0494L | £623 | 1 | £623 | |
| NEBNext® UltraII™ End-prep | NEB | E7546L | £729 | 1 | £729 | |
| NEBNext® UltraII™ Ligation Module | NEB | E7595L | £1,204 | 1 | £1,204 | |
| NEBNext® Quick Ligation Module | NEB | E6056S | £317 | 1 | £317 | |
| Aline PCRCLEAN DX 50ml | Aline | AL-AC1003-50 | £325 | 1 | £325 | |
| Ligation Sequencing Kit 1D | Nanopore | SQK-LSK109 | £470 | 1 | £470 | |
| Native Barcoding Expansion Kit 1-12 | Nanopore | EXP-NBD104 | £235 | 1 | £235 | |
| Native Barcoding Expansion Kit 13-24 | Nanopore | EXP-NBD114 | £235 | 1 | £235 | |
| Flow Cell Priming Kit | Nanopore | EXP-FLP002 | £30 | 2 | £60 | |
| Flow Cell Wash Kit | Nanopore | EXP-WSH003 | £90 | 2 | £180 | |
| R9.4.1 flow cells | Nanopore | £700 | 2 | £1,400 | ||
| QuantiFluor ONE dsDNA System | Promega | E4870 | £187 | 1 | £187 | |
| TOTAL COST | £6,890 |
Equipment and Containment
| Product | Supplier | Cat No. | Unit Cost | # Req | Cost | |
|---|---|---|---|---|---|---|
| 1000µL pipettes | Gilson | 10387322 | £210 | 2 | £420 | |
| 100µL pipettes | Gilson | 10442412 | £210 | 2 | £420 | |
| 10µL pipettes | Gilson | 10236072 | £210 | 2 | £420 | |
| 2µL pipettes | Gilson | 10082012 | £210 | 2 | £420 | |
| Sprout Microfuge (12V) | PhilipHarris | B8R04574 | £170 | 2 | £340 | |
| PV-1 Mini Vortex | FisherSci | PV-1 | £150 | 2 | £300 | |
| Portable Heat Block | Mobitec | Pad3-H-CB | £310 | 1 | £310 | |
| DynaMag-2 magnetic rack | Thermo | 12321D | £540 | 1 | £540 | |
| MiniPCR™ Mini16 | Cambio | Mp-QP-1016-01 | £675 | 2 | £1,350 | |
| Quantus Fluorometer | Promega | E6150 | £1,236 | 1 | £1,236 | |
| PCR Workstation | StarLabs | E2396-9693 | £20 | 3 | £60 | |
| Collapsible Tube racks (large x5) | FisherSci | 11710634 | £50 | 1 | £50 | |
| Cold Racks (0.2-0.5mL) | Sigma | Z606634-1EA | £50 | 2 | £100 | |
| Lab Mats | StarLab | E3860-0061 | £25 | 2 | £50 | |
| USB Lights | Amazon | Jebsen-T05 | £20 | 2 | £40 | |
| Portable UV Lamps | Amazon | B078LSR3MG | £20 | 2 | £40 | |
| Portable glove box | Amazon | B01B1MGRR0 | £70 | 2 | £40 | |
| TOTAL COST | £6,136 |
Consumables and Disposables
| Product (Number) | Supplier | Cat No. | Unit Cost | # Req | Cost | |
|---|---|---|---|---|---|---|
| Stacked 1000µL Filter Tips (500) | FisherSci | 11977724 | £70 | 2 | £140 | |
| Stacked 100µL Filter Tips (1000) | FisherSci | 11947724 | £60 | 1 | £60 | |
| Stacked 10µL Filter Tips (1000) | FisherSci | 11907724 | £60 | 1 | £60 | |
| 1.5mL Eppendorf Tubes (500) | StarLab | I1415-2500 | £20 | 2 | £40 | |
| 8-strip PCR tubes with Individual Caps, 0.2 ml (1000) | StarLab | I1402-2900 | £115 | 1 | £115 | |
| 0.5 ml thin-walled tubes (1000) | Axygen | PCR-05-C | £40 | 1 | £40 | |
| MyGo PCR Tubes (500) | IT-IS | SKU7688 | £85 | 1 | £85 | |
| BioBIn® Disposal Bins (100) | FisherSci | 10394373 | £121 | 1 | £121 | |
| Gloves (S/M/L, 100) | StarLab | SG-P-X | £5 | 2 | £10 | |
| Paper Towels | Generic | 4 | N/A | |||
| Decon Wipes (240) | MediPal | PMC6038 | £6 | 2 | £12 | |
| Back-tie Lab Gowns (45G) | VWR | 113-8316 | £56 | 10 | £56 | |
| RNAse Away (250mL) | Thermo | 10328011 | £60 | 2 | £120 | |
| DNA Away (250mL) | Thermo | 7010PK | £20 | 2 | £40 | |
| TOTAL COST | £887 |
Cold Chain, Power and Storage
| Product (Number) | Supplier | Cat No. | Unit Cost | # Req | Cost | |
|---|---|---|---|---|---|---|
| Flat dehydrated cold sheets (ThermaFreeze) | Amazon | B003ET2G74 | £15 | 10 | £150 | |
| Thermos Radiance Cooler Bag | Amazon | B00BHW5B4U | £10 | 4 | £40 | |
| Portable Fridge Bag | Amazon | B008N5UU3G | £70 | 1 | £70 | |
| Credo Cube Series 20M 4L | Pelican | Contact Supp. | N/A | |||
| PowerAdd Pilot Pro2 85Wh LiION 9-20V | Amazon | B013HXKZYW | £80 | 2 | £160 | |
| Solar Charger, 85Wh, LiION 5-20V | Amazon | B00AE9FBCO | £85 | 2 | £170 | |
| Goal Zero Yeti 150 Portable Power Station | GoalZero | SKU22004 | £200 | 1 | £200 | |
| Goal Zero Nomad 28 Plus Solar Panel | Goal Zero | SKU11805 | £250 | 1 | £250 | |
| Surge Protector Extension (4-plug) | Belkin | B0002AG0F2 | £6 | 3 | £18 | |
| SKROSS Universal Mini Travel Adaptor | Amazon | B00OHSWG6C | £10 | 2 | £20 | |
| Peli Air 1615 Waterproof Equipment Case | RS Comp. | 121-8893 | £550 | 1 | £550 | |
| Eagle Creek Cargo Hauler, 120L | Amazon | B01HIGO3OC | £150 | 1 | £150 | |
| BESTEK 200W Power Inverter DC 12V | Amazon | B01N2P9V64 | £30 | 1 | £30 | |
| Samsung Portable SSD T5 1TB | Amazon | B074M774TW | £320 | 2 | £640 | |
| Seagate® 5TB External HD | Amazon | B01M058Q39 | £119 | 2 | £238 | |
| TOTAL COST | £2,686 |
