Ligation sequencing DNA V14 - automated Tecan DreamPrep NGS (SQK-LSK114-XL)
PromethION: Protocol
Ligation sequencing DNA V14 - automated Tecan DreamPrep NGS (SQK-LSK114-XL) V DTD_9181_v114_revK_22Feb2023
- This protocol uses genomic DNA
- Hands-on setup time ~30 minutes
- Automated run time ~2 hours 40 minutes
- Throughput: Process 8–96 samples for singleplex library loading
- Compatible with R10.4.1 Flow Cells
For Research Use Only
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Automated library preparation
- 4. Library preparation
- 5. Priming and loading the PromethION Flow Cell
- 6. Unloading the Tecan DreamPrep NGS worktable
Secuenciación y análisis
Troubleshooting
Disclaimers
Descripción general
- This protocol uses genomic DNA
- Hands-on setup time ~30 minutes
- Automated run time ~2 hours 40 minutes
- Throughput: Process 8–96 samples for singleplex library loading
- Compatible with R10.4.1 Flow Cells
For Research Use Only
1. Overview of the protocol
IMPORTANTE
This protocol describes the automated workflow using the Ligation Sequencing Kit XL (SQK-LSK114-XL).
All images and information reflect the use of the use of SQK-LSK114-XL.
For more information on compatibilities and performing an automated library preparation with previous iterations of the Ligation Sequencing Kit, please contact us on our website by following the link.
Ligation Sequencing Kit XL V14 features
This kit is recommended for users who:
- Would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid-handling robot
- Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above
- Require control over read length
- Would like to utilise upstream processes such as size selection, whole genome amplification, or enrichment for long reads
IMPORTANTE
Optional fragmentation and size selection
By default, the protocol contains no DNA fragmentation step, however in some cases it may be advantageous to fragment your sample. For example, when working with lower amounts of input gDNA (100 ng – 500 ng), fragmentation will increase the number of DNA molecules and therefore increase throughput. Instructions are available in the DNA Fragmentation section of Extraction methods.
Additionally, we offer several options for size-selecting your DNA sample to enrich for long fragments - instructions are available in the Size Selection section of Extraction methods.
IMPORTANTE
Kit 14 sequencing and duplex basecalling info sheet
The Kit 14 chemistry is a new development from Oxford Nanopore Technologies with improved duplex basecalling, which requires a different set of tools. For more information, please see the Kit 14 sequencing and duplex basecalling info sheet. We strongly recommend that you read it before proceeding with Kit 14 chemistry sequencing experiments and basecalling duplex data.
Introduction to the automated Ligation Sequencing protocol for DNA
This protocol describes how to carry out sequencing of a DNA sample using the Ligation Sequencing Kit XL (SQK-LSK114-XL).
We have developed this automated protocol on the Tecan® DreamPrep® NGS liquid handling robot. This library preparation protocol is fully automated, except for the sample quantification steps and loading of the system.
Please note that this method is intended for research use only.
It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
To efficiently load multiple PromethION Flow Cells, we recommend using the Loading multiple PromethION Flow Cells protocol as a guideline.
Steps in the sequencing workflow:
#### Prepare for your experiment You will need to:
Before starting - Manual steps:
- Extract your DNA and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment, primed liquid-handling robot and third-party reagents
- Download the software for acquiring and analysing your data
- Check your flow cells to ensure they have enough pores for a good sequencing run
Prepare your library
You will need to:
Automated steps:
- Repair the DNA and prepare the DNA ends for adapter attachment
- Attach sequencing adapters supplied in the kit to the DNA ends
__Manual steps:__ - Quantify your DNA library as a quality control
- Prime the flow cell and load your DNA library into the flow cell
Overview of library preparation workflow:
Note: Timings are dependent on number of samples and include hands on time, such as deck loading and sample quantification
Sequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software which will collect raw data from the device and convert it into basecalled reads
Timings
Note: Timings are approximate and subject to change with updates.
| Process | X8 samples | X24 samples | X48 samples | X96 samples | Hands-on time |
|---|---|---|---|---|---|
| Deck set-up and master mix preparation | ~30 minutes | ||||
| Automated DNA repair, end-prep and clean-up | 1 hour | 1 hour | 1 hour 5 minutes | 1 hour 10 minutes | |
| Automated adapter ligation and clean-up | 1 hour 30 minutes | 1 hour 30 minutes | 1 hour 35 minutes | 1 hour 35 minutes | |
| Quantification | ~10 minutes | ||||
| Total | 2 hours 30 minutes | 2 hours 30 minutes | 2 hours 40 minutes | 2 hours 45 minutes | ~40 minutes |
Sequencing run set-up and flow cell loading timings are variable depending on the number of samples and user experience.
IMPORTANTE
Compatibility of this protocol
This protocol should only be used in combination with:
- Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
- R10.4.1 flow cells (FLO-PRO114M)
- Flow Cell Wash Kit XL (EXP-WSH004-XL)
2. Equipment and consumables
Material
- 1 µg (o 100-200 fmol) de ADN genómico de alto peso molecular
- o 100+ ng de ADN genómico de alto peso molecular (si se fragmenta el ADN).
- Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
Consumibles
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB E7180S or E7180L) (módulo de acompañamiento NEBNext de secuenciación por ligación para Oxford Nanopore Technologies®) Como alternativa, se pueden utilizar los siguientes productos de NEBNext®:
- NEBNext FFPE Repair Mix (NEB M6630) (mezcla de reparación de ADN)
- NEBNext Ultra II End Repair/dA-tailing Module (NEB E7546) (Módulo de reparación de extremos/Adición de dA)
- NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
- Etanol al 80 % recién preparado con agua sin nucleasas
- Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted, red/clear (Bio-Rad™, cat # HSP9611)
- Thermo Scientific™ Abgene™ 96 Well 1.2 ml Polypropylene Deepwell Storage Plate (Thermo Scientific, cat # AB1127)
- 1000 µl Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057817)
- 200 ul Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057815)
- 50 ul Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057813)
- Small SBS Box to place conductive tips & refill, compatible with 10uL, 50uL, 200uL tips (Tecan , cat# 30058506)
- Big SBS Box to place conductive tips & refill, compatible with 1000uL tips (Tecan , cat# 30058507)
- 150 µl Disposable Tips - MultiChannel Arm™ 384/96 - Filtered, Sterile, Single Stack (Tecan, cat # 30038618)
- 50 µl Disposable Tips - MultiChannel Arm 384/96 - Filtered, Sterile, Single Stack (Tecan, cat # 30038608)
- 100 ml disposable trough (Tecan, cat # 10613049)
- 25 ml disposable trough (Tecan, cat # 30055743)
- Arched Auto-Sealing Lids with Wide Tabs for PCR Plates (Bio-Rad™, cat # MSL2032 or equivalent)
- Sarstedt Inc Screw Cap Micro tube 2 ml, sterile (Sarstedt™, cat # 72.694.321)
- Tubos de 1,5 ml Eppendorf DNA LoBind
- 2 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
- Low-lint scientific wipes (e.g. Kimberly-Clark™, cat # 7552 or equivalent)
- Double distilled water (ddH2O) (e.g. ThermoFisher, cat # 11983084)
- Freshly prepared ≥80% ethanol in nuclease-free or double distilled water, for cleaning
- ~10% Bleach (or equivalent): Thermo Scientific Alfa Aesar Sodium hypochlorite, 11-15% available chlorine, (e.g. ThermoFisher, cat # 15429019)
Instrumental
- Cubeta con hielo
- Mezclador vórtex
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Tecan DreamPrep NGS workstation with full configuration
Equipo opcional
- Bioanalizador Agilent (o equivalente)
- Qubit fluorometer plate reader (or equivalent for QC check)
Ajuste la cantidad de muestra en función de la longitud de la muestra de ADN inicial:
| Longitud de fragmentos | Cantidad de muestra inicial |
|---|---|
| Muy cortos (<1 kb) | 200 fmol |
| Cortos (1-10 kb) | 100–200 fmol |
| Largos (>10 kb) | 1 µg |
Para más información acerca de las cantidades de muestra inicial y de carga de las celdas de flujo en los protocolos de secuenciación por ligación, consulte este documento técnico
Input DNA
How to QC your input DNA
It is important to use a plate reader to ensure the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.
Tecan DreamPrep NGS
This method has been tested and validated using the Tecan DreamPrep NGS including an on deck thermal cycler (ODTC). An option to not use the ODTC is available in the method. This protocol will require installation by a Tecan Filed Application Specialist, please contact your Tecan representative for further details.
Tecan DreamPrep NGS worktable layout
After having the system installed according to specifications, users will need to load the automated workstation before running the protocol.
Please always refer to the latest instructions on how to load the worktable, which are displayed and explained in the touchscreen display (with TouchTools™).
Examples of how to load the system are displayed from steps 11 to 20, which can be found in the library preparation section of the protocol.
Please also find below reference images for the workbench layouts:
Trough mounting sites:
Hotel sites:
NEBNext® Companion Module para secuenciación por ligación, de Oxford Nanopore Technologies®
A los clientes que no conozcan la secuenciación por nanoporos, se les recomienda que adquieran el módulo de acompañamiento NEBNext® Ligation Sequencing para Oxford Nanopore Technologies® (NEB E7180S o E7180L), que contiene todos los reactivos NEB necesarios para usar con el kit Ligation Sequencing Kit.
Nótese que en los protocolos con amplicones no es necesario usar ni la mezcla de reparación de ADN NEBNext FFPE DNA Repair Mix, ni el tampón de reparación de ADN NEBNext FFPE DNA Repair Buffer.
Reactivos de otros fabricantes
Oxford Nanopore Technologies ha probado y recomienda el uso de todos los reactivos de otros fabricantes citados en este protocolo. No se han evaluado otras alternativas.
Recomendamos preparar estos reactivos siguiendo las instrucciones del fabricante.
IMPORTANTE
A fin de garantizar un elevado rendimiento de ligación del adaptador Ligation Adapter (LA), recomendamos el uso del tampón Ligation Buffer (LNB) incluido en el kit Ligation Sequencing Kit V14, en lugar del tampón de ligasa de otros fabricantes.
IMPORTANTE
El adaptador incluido en este kit, Ligation Adapter (LA), no es intercambiable con otros adaptadores de secuenciación.
Consumables and reagent quantities required:
| Consumables | X8 samples | X24 samples | X48 samples | X96 samples |
|---|---|---|---|---|
| Tecan 1000 µl Flexible Channel Arm filter tips | 49 tips | 50 tips | 51 tips | 53 tips |
| Tecan 200 µl Flexible Channel Arm filter tips | 33 tips | 32 tips | 32 tips | 32 tips |
| Tecan 50 µl Flexible Channel Arm filter tips | 16 tips | 16 tips | 16 tips | 16 tips |
| Tecan 150 µl MultiChannel Arm 384/96 filter tips | 1 box | 3 boxes | 5 boxes | 10 boxes |
| Tecan 50 µl MultiChannel Arm 384/96 filter tips | 1 box | 2 boxes | 3 boxes | 5 boxes |
| Bio-Rad Hard-Shell® 96-Well PCR Plate | 8 | 8 | 8 | 8 |
| Abgene™ 96 Well 1.2mL Polypropylene Deepwell Storage Plate | 1 | 1 | 1 | 1 |
| Tecan 25 ml disposable trough | 4 | 4 | 4 | 3 |
| Tecan 100 ml disposable trough | 1 | 1 | 1 | 2 |
| ODTC plate lid | 1 | 1 | 1 | 1 |
| Sarstedt Inc Screw Cap Micro tube 2 ml | 2 | 2 | 3 | 5 |
| Reagents/kits | X8 samples | X24 samples | X48 samples | X96 samples |
|---|---|---|---|---|
| 80% ethanol | 10 ml | 16 ml | 24 ml | 42 ml |
| Nuclease-free water | 3.5 ml | 4.6 ml | 6.3 ml | 9.5 ml |
| AMPure XP Beads | 3 ml | 4.9 ml | 7.8 ml | 13.5 ml |
| Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) | 1 kit | 1 kit | 1 kit | 2 kits |
| NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180L) | 1 kit | 1 kit | 1 kit | 1 kit |
| NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S) | - | - | - | 1 kit |
| Alternative to the NEBNext Companion Module, individual reagents can be bought as seen below: | - | - | - | - |
| NEBNext FFPE DNA Repair Mix (Cat# M6630L) | 1 kit | 1 kit | 1 kit | 2 kits |
| NEBNext Ultra II End Repair/dA-Tailing Module (Cat# E7546L) | 1 kit | 1 kit | 1 kits | 2 kits |
| NEBNext Quick Ligation Module (Cat# E6056L) | 1 kit | 1 kit | 1 kit | 2 kits |
| NEBNext Quick Ligation Module (Cat# E6056S) | - | - | 1 kit | 1 kit |
| Note: These are the number of kits required for one run through for the selected number of samples. |
Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) contents
| Name | Acronym | Vial colour | Number of vials | Fill volume per vial (µl) |
|---|---|---|---|---|
| DNA Control Strand | DCS | Yellow | 1 | 100 |
| Ligation Adapter | LA | Green | 1 | 320 |
| Ligation Buffer | LNB | White | 1 | 1,500 |
| Elution Buffer | EB | White cap, black strip label | 1 | 10,000 |
| Long Fragment Buffer | LFB | White cap, orange strip label | 2 | 20,000 |
| Short Fragment Buffer | SFB | White cap, blue strip label | 2 | 20,000 |
| Library Beads | LIB | Pink | 2 | 1,800 |
| Library Solution | LIS | White cap, pink label | 2 | 1,800 |
| Sequencing Buffer | SB | Red | 3 | 1,700 |
| Flow Cell Flush | FCF | Clear | 4 | 15,500 |
| Flow Cell Tether | FCT | Purple | 1 | 1,600 |
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.
3. Computer requirements and software
PromethION 24/48 IT requirements
The PromethION device contains all the hardware required to control up to 24 (for the P24 model) or 48 (for the P48 model) sequencing experiments and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the PromethION IT requirements document.
PromethION 2 Solo IT requirements
The PromethION 2 (P2) Solo is a device which directly connects into a GridION Mk1 or a stand-alone computer that meets the miminum specifications for real-time data streaming and analysis. Up to two PromethION flow cells can be can be run and each is independently addressable, meaning experiments can be run concurrently or individually. For information on the computer IT requirements, please see the PromethION 2 Solo IT requirements document.
Software for nanopore sequencing
MinKNOW
The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.
For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.
EPI2ME (optional)
The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.
For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform protocol.
Verificar la celda de flujo
Antes de empezar el experimento de secuenciación, recomendamos verificar el número de poros disponibles, presentes en la celda de flujo. La comprobación deberá realizarse en las primeras 12 semanas desde su adquisición, si se trata de celdas de flujo MinION, GridION o PromethION, y en las primeras cuatro semanas tras la compra de celdas de flujo Flongle. Oxford Nanopore Technologies sustituirá cualquier celda de flujo con un número de poros inferior al indicado en la tabla siguiente, siempre y cuando el resultado se notifique dentro de los dos días siguientes a la comprobación y se hayan seguido las instrucciones de almacenamiento. Para verificar la celda de flujo, siga las instrucciones del documento Flow Cell Check.
| Celda de flujo | Número mínimo de poros activos cubierto por la garantía |
|---|---|
| Flongle | 50 |
| MinION/GridION | 800 |
| PromethION | 5000 |
4. Library preparation
Material
- 1 µg (o 100-200 fmol) de ADN genómico de alto peso molecular
- Ligation Adapter (LA) (adaptador de ligación)
- Ligation Buffer (LNB) (tampón de ligación) del kit Ligation Sequencing Kit
- Long Fragment Buffer (LFB) (tampón para fragmentos largos)
- Short Fragment Buffer (SFB)
- Elution Buffer from the Oxford Nanopore kit (EB)
Consumibles
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (cat # E7180S or E7180L). Alternatively, you can use the NEBNext® products below:
- NEBNext FFPE DNA Repair Mix (NEB M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB E7546)
- NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Etanol al 80 % recién preparado con agua sin nucleasas
- Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted, red/clear (Bio-Rad™, cat # HSP9611)
- Thermo Scientific™ Abgene™ 96 Well 1.2 ml Polypropylene Deepwell Storage Plate (Thermo Scientific, cat # AB1127)
- 5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- 1000 µl Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057817)
- 200 ul Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057815)
- 50 ul Disposable Conductive Tips - Liquid Handling Flexible Channel Arm - Filtered, Pure, ANSI/SLAS-format box (same as SBS) (Tecan , cat# 30057813)
- Small SBS Box to place conductive tips & refill, compatible with 10uL, 50uL, 200uL tips (Tecan , cat# 30058506)
- Big SBS Box to place conductive tips & refill, compatible with 1000uL tips (Tecan , cat# 30058507)
- 150 µl Disposable Tips - MultiChannel Arm™ 384/96 - Filtered, Sterile, Single Stack (Tecan, cat # 30038618)
- 50 µl Disposable Tips - MultiChannel Arm 384/96 - Filtered, Sterile, Single Stack (Tecan, cat # 30038608)
- 100 ml disposable trough (Tecan, cat # 10613049)
- 25 ml disposable trough (Tecan, cat # 30055743)
- Sarstedt Inc Screw Cap Micro tube 2 ml, sterile (Sarstedt™, cat # 72.694.321)
Instrumental
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- Pipeta y puntas P10
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Mezclador vórtex
Equipo opcional
- Qubit fluorometer plate reader (or equivalent for QC check)
IMPORTANTE
Optional fragmentation and size selection
By default, the protocol contains no DNA fragmentation step, however in some cases it may be advantageous to fragment your sample. For example, when working with lower amounts of input gDNA (100 ng – 500 ng), fragmentation will increase the number of DNA molecules and therefore increase throughput. Instructions are available in the DNA Fragmentation section of Extraction methods.
Additionally, we offer several options for size-selecting your DNA sample to enrich for long fragments - instructions are available in the Size Selection section of Extraction methods.
Preparar los reactivos NEBNext FFPE DNA Repair Mix y NEBNext Ultra II End Repair / dA-tailing Module siguiendo las instrucciones del fabricante y poner en hielo.
Para obtener un rendimiento óptimo, NEB recomienda lo siguiente:
Descongelar todos los reactivos en hielo.
Golpear suavemente los tubos de reactivos con el índice o invertirlos, para asegurarse de que estén bien mezclados.
Nota: No mezclar en vórtex las mezclas FFPE DNA Repair Mix, ni Ultra II End Prep Enzyme Mix.Centrifugar los tubos antes de abrirlos.
Los tampones Ultra II End Prep Buffer y FFPE DNA Repair Buffer pueden tener un poco de precipitado. Dejar que la mezcla alcance la temperatura ambiente y mezclar pipeteando varias veces para romper el precipitado; para solubilizarlo, agitar el tubo en vórtex durante 30 s.
Nota: Es importante mezclar bien los tampones mediante vórtex.El tampón FFPE DNA Repair Buffer puede tener un matiz amarillo; no importa si está así; se puede utilizar.
Switch on the Tecan DreamPrep NGS robot and open the Fluent Control software on the computer. Follow the recommended specifications to initiate the DreamPrep NGS.
IMPORTANTE
Perform the 'Daily System Care' method to prepare the instrument before the first run of the day.
Users will have access to the 'Main screen' of TouchTools™, which allows interaction with the DreamPrep NGS system. Select 'Method Starter'.
In the 'Method Starter' folder, select the Ligation sequencing program and click 'Ok'.
Click the 'start button' in the middle of the screen to start the run.
During the run set-up on the Tecan DreamPrep NGS, the workstation will perform automated checks and set-up for the ODTC.
During the run set-up, messages will pop-up regarding the ODTC set-up.
Allow these to complete and continue your run set-up as normal.
When you see the 'Welcome to the ONT Ligation Sequencing protocol by Tecan' page, click 'Continue'.
Set the 'User defined variables' and click on 'Next page' to proceed.
Note: Any number of samples between 8-96 can be processed using this protocol.
Set the 'User inputs' and click on 'Next page' to proceed.
- Please note that if removing the sample plate for off-deck storage, user interaction is required approximately 10 minutes after starting the run.
- We recommend room temperature for the majority of users. However, 37°C can be beneficial for recovery of longer DNA strands.
- The MinION option is valid for both the MinION and GridION device.
MEDIDA OPCIONAL
Reminder: If selecting 'YES' on 'Store Sample plate off-deck after use', user interaction will be required 10 minutes after starting the run. Click 'Next Page' to proceed.
Select if you would like to maintain the standard LSK volumes and timers as recommended by ONT and click on 'Next page' to proceed.
Note: Please note only the ONT recommended settings have been verified.
IMPORTANTE
We highly recommend using default settings developed by Oxford Nanopore Technologies.
Only personalise run settings if you are an experienced user. Any deviations from the default settings have not been qualified and are done at the operators risk.
MEDIDA OPCIONAL
If using personalised volume and timer inputs:
- Select 'No' under 'Use standard volume and timers'.
Note: All categories will be autofilled with the recommended settings. These can be edited for personal requirements.
- Enter the desired reagent volumes for each sample in the 'End-repair' section I and II.
- Enter the desired length for each process in the 'End-repair' section of the protocol.
- Enter the desired reagent volumes for each sample in the 'Adapter ligation' section I and II.
- Enter the desired length for each process in the 'End-repair' section of the protocol.
Review the run variables and click 'Confirm':
Follow the on-screen directions to load the 150 µl filtered tips for the Multichannel Arm 384/96 onto the worktable:
- The required loading position for each box will flash to indicate where to place the labware.
- Please load full tip boxes only, partially full boxes of filtered tips for the Multichannel Arm 384/96 are currently not supported.
- Click 'Approve' after each addition of labware to proceed to the next box.
- After loading all of the required labware, close the front safety shield and click 'Next Page' to proceed.
Follow the on-screen directions to load the 50 µl filtered tips for the Multichannel Arm 384/96 onto the worktable:
- The required loading position for each box will flash to indicate where to place the labware.
- Please load full tip boxes only, partially full boxes of filtered tips for the Multichannel Arm 384/96 are currently not supported.
- Click 'Approve' after each addition of labware to proceed to the next box.
- After loading all of the required labware, close the front safety shield and click 'Next Page' to proceed.
Follow the on-screen directions to load the 1000 µl Flexible Channel Arm filtered tips onto the worktable:
- The required loading position for each box will flash to indicate where to place the labware.
- Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
- Click 'Approve' after each addition of labware to proceed to the next box.
Follow the on-screen directions to load the 200 µl Flexible Channel Arm filtered tips onto the worktable:
- The required loading position for each box will flash to indicate where to place the labware.
- Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
- Click 'Approve' after each addition of labware to proceed to the next box.
Follow the on-screen directions to load the 50 µl Flexible Channel Arm filtered tips onto the worktable:
- The required loading position for each box will flash to indicate where to place the labware.
- Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
- Click 'Approve' after each addition of labware to proceed to the next box.
- After loading all of the required labware, click 'Next Page' to proceed.
Follow the on-screen directions to load the metal lid for ODTC on to the worktable:
- The required loading position will flash to indicate where to place the labware.
- Click 'Approve' after the addition of the metal lid for ODTC to proceed.
Loading the metal lid for ODTC:
Note: Take care to position the metal lid centrally in the recess. Incorrect positioning of the metal lid can lead to run error.
Follow the on-screen directions to load the Waste plate on to the worktable:
- The required loading position will flash to indicate where to place the labware.
- After loading the Waste plate, click 'Next Page'.
Loading the Waste Plate:
Follow the on-screen directions to load the reaction plates onto the worktable:
Incorrect positioning of the reaction plates will result in incorrect sample tracking throughout the run. Please ensure the reaction plates are placed on the worktable in the correct orientation:
For the reaction plate(s) loaded in the 'Hotel': The lettered well markers (A-H) should be positioned towards the back of the worktable and the numbered well markers (1-12) should be positioned facing the right.
For the reaction plate(s) loaded onto the worktable: Follow standard plate orientation, with the lettered well markers (A-H) positioned to the left and the numbered well markers (1-12) positioned to towards the back of the worktable.
__Note:__ We recommend all plates are labelled before placing on the worktable to ensure correct plate tracking.
The required loading position for each plate will flash on the on-screen display to indicate where to place the labware.
Click 'Approve' after each addition of labware to proceed.
After loading all of the required labware, click 'Next Page'.
Follow the on-screen directions to load the Bead plate, the Elution buffer plate and Ethanol plate on to the worktable:
- The required loading position will flash to indicate where to place the labware.
- Click 'Approve' after each addition of labware to proceed.
- After loading all of the required labware, click 'Next Page'.
Note: We recommend all plates are labelled before placing on the worktable to ensure correct plate tracking.
Loading the 'Bead plate':
Loading the 'Elution buffer plate':
Loading the 'Ethanol plate':
Close the front safety shield of the Tecan DreamPrep NGS while you prepare the reaction master mixes off-deck.
Prepare the End-prep (EP) master mix in a 2 ml Sarstedt tube with the following reagents according to the Tecan DreamPrep NGS user interface. Click 'OK' and 'Continue' to proceed.
We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.
- Ensure the master mix is homogenous by pipette mixing.
- Avoid the introduction of air bubbles while preparing the master mix.
- Avoid leaving droplets on the tube wall.
- Briefly spin down the End-prep (EP) master mix to ensure all the liquid is at the bottom of the Sarstedt tube.
Reagent volumes for all sample numbers:
| Reagent | Volume per sample | Volume X8 samples | Volume X24 samples | Volume X48 samples | Volume X96 samples |
|---|---|---|---|---|---|
| NEBNext FFPE DNA Repair Buffer | 3.5 µl | 36.4 µl | 109.2 µl | 201.6 µl | 369.6 µl |
| NEBNext FFPE DNA Repair Mix | 2 µl | 20.8 µl | 62.4 µl | 115.2 µl | 211.2 µl |
| Ultra II End-prep reaction buffer | 3.5 µl | 36.4 µl | 109.2 µl | 201.6 µl | 369.6 µl |
| Ultra II End-prep enzyme mix | 3 µl | 31.2 µl | 93.6 µl | 172.8 µl | 316.8 µl |
| Total | 12 µl | 124.8 µl | 374.4 µl | 691.2 µl | 1267.2 µl |
Note: Reagent volumes will vary in accordance with the number of samples selected for processing (8-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes. Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.
Prepare the Adapter-ligation (AL) master mix directly into the 2 ml Sarstedt tube(s) with the following reagents according to the Tecan DreamPrep NGS user interface. Click 'OK' and 'Continue' to proceed.
We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.
- Ensure the master mix is homogenous by pipette mixing.
- Avoid the introduction of air bubbles while preparing the Adapter-ligation (AL) master mix.
- Avoid leaving droplets on the tube wall.
- Briefly spin down the Adapter-ligation (AL) master mix to ensure all the liquid is at the bottom of the Sarstedt tube(s).
Reagent volumes for preparation in each tube for all sample numbers:
| Reagent | Volume per sample | Volume X8 samples | Volume X24 samples | Volume X48 samples | Volume X96 samples |
|---|---|---|---|---|---|
| Number of tubes to prepare | - | 1 | 1 | 2 | 4 |
| Ligation Buffer (LNB) | 25 µl | 260 µl | 780 µl | 660 µl | 660 µl |
| NEBNext Quick T4 DNA Ligase | 10 µl | 104 µl | 312 µl | 264 µl | 264 µl |
| Ligation Adapter (LA) | 5 µl | 52 µl | 156 µl | 132 µl | 132 µl |
| Total volume in each tube | - | 416 µl | 1,248 µl | 1,056 µl | 1,056 µl |
| Total volume prepared | - | 416 µl | 1,248 µl | 2,112 µl | 4,224 µl |
| Note: Reagent volumes will vary in accordance with the number of samples selected for processing (8-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes and split accordingly across the Sarstedt tube(s). | |||||
| Volumes indicated in the table and the on-screen display will include the necessary dead volume excess. |
Load the DNA repair and end-prep master mix and Adapter ligation master mix prepared above in the 2 ml Sarstedt tubes into the required positions in the POGO tube holder by following the on-screen instructions.
- Ensure the master mixes are thoroughly mixed before loading.
- You will need to select each loading position using the Tecan's TouchTools touchscreen display.
- Follow the instructions on the display for each reagent, ensuring the fill volume for each tube is correct.
- Ensure the reagents have been added to all of the required positions before proceeding.
- Click 'Confirm' to proceed.
On-screen abbreviation glossary:
- End-prep master mix – EP
- Adapter ligation master mix – AL
IMPORTANTE
Please ensure the liquid is evenly distributed across the troughs.
Gently tilt liquid in the trough backwards and forward a few times to generate a wet surface and allow the full volume to be evenly distributed.
Uneven distribution of the volume in the trough can be detrimental to the run.
CONSEJO
Trough mounts and loading:
Trough mount sites:
Trough loading:
For the 100 ml troughs, dispense the required reagent volume directly into the trough and load on the the worktable. The 100 ml trough use will be reflected on the screen display with the following image:
For the 25 ml troughs, dispense the required reagent volume into the trough. You will then need to insert the 25 ml trough into a 100 ml trough to act as a holder or use a re-useable metal insert (for more information on additional equipment contact your Tecan representative). The modified 25 ml trough containing the reagent will then be loaded into the worktable. This input will be reflected on the screen display with the following image:
IMPORTANTE
La fase de lavados tras la ligación de los adaptadores está diseñada para enriquecer los fragmentos de ADN de >3 kb o para purificar todos los fragmentos por igual, según el tampón que se utilice -Long Fragment Buffer (LFB) o Short Fragment Buffer (SFB).
Para enriquecer fragmentos de ADN de 3 kb o mayores, utilizar el tampón para fragmentos largos, Long Fragment Buffer (LFB).
Para conservar fragmentos de ADN de todos los tamaños, utilizar el tampón para fragmentos cortos, Short Fragment Buffer (SFB).
Load the troughs with their relevant reagents into the worktable by following the on-screen instructions.
- Ensure all the reagents have been thoroughly mixed by vortexing before dispensing into the troughs.
- The required loading position will flash to indicate where to insert the trough.
- Click 'Approve' after each addition to proceed.
- After loading all of the troughs, click 'Next Page'.
__Note:__ Follow the on-screen instructions for the reagent fill volume and the required trough to use.
- Site 2: Trough with fresh 80% ethanol.
- Site 3: Trough with AMPure XP Beads.
- Site 5: Trough with Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB), depending on use.
- Site 6: Trough with Elution Buffer (EB).
- Site 7: Trough with nuclease-free water.
Close the front safety shield of the Tecan DreamPrep NGS while you prepare the sample plate off-deck.
Follow the on-screen directions to load the sample plate on to the worktable:
- Quantify your sample input using a Qubit fluorometer (or equivalent).
- Per sample, transfer 1 μg (or 100-200 fmol) of input DNA into a well of the input plate.
- Adjust the volume to 60 μl with nuclease-free water.
- Mix thoroughly by pipetting.
- Spin down briefly in a microfuge.
- The required loading position will flash to indicate where to place the labware.
- Click 'Approve' after the addition of labware to proceed.
- After loading all of the required labware, click 'Next Page'.
MEDIDA OPCIONAL
If removing the sample plate for off-deck storage, user interaction is required approximately 10 minutes after starting the run.
Close the front safety shield of the Tecan DreamPrep NGS before starting your run and click 'Continue'.
Click 'Confirm' and 'Continue' to start the automated library preparation.
CONSEJO
Once the run has finished on the Tecan DreamPrep NGS remove your elution plate as soon as possible from the worktable to avoid evaporation.
We do not recommend running the liquid handling robot overnight as leaving the eluted library plate unsealed can lead to evaporation of the library product.
Please refer to the 'Timings' table found in the overview of the protocol for guidance.
Remove the plate containing the eluted libraries from the Tecan DreamPrep NGS deck.
Quantify 1 µl of each eluted sample from the output plate using a Qubit fluorometer plate reader off deck.
FIN DEL PROCESO
Seal the plate and store on ice until ready to prepare the library/libraries and load onto the flow cell.
We do not recommend running the liquid handling robot overnight as the plate must be sealed and stored on ice as soon as library preparation is finished.
Depending on your DNA library fragment size, prepare your final library in 32 µl of Elution Buffer (EB).
| Fragment library length | Flow cell loading amount |
|---|---|
| Very short (<1 kb) | 100 fmol |
| Short (1-10 kb) | 35–50 fmol |
| Long (>10 kb) | 300 ng |
Note: If the library yields are below the input recommendations, load the entire library.
If required, we recommend using a mass to mol calculator such as the NEB calculator.
IMPORTANTE
Recomendamos cargar una de las siguientes cantidades de biblioteca, en la celda de flujo R10.4.1.
Para obtener un rendimiento elevado de datos simplex, tome 35-50 fmol de biblioteca. De este modo se garantiza una elevada ocupación de poros >95 %. Aquí puede consultar cómo calcular la ocupación de poros.
Para obtener datos duplex, cargue 10-20 fmol de biblioteca. Cargar más de 20 fmol de ADN puede reducir la velocidad de recogida de lecturas duplex.
CONSEJO
Library storage recommendations
We recommend storing libraries at 4°C for short term storage or repeated use, for example, re-loading flow cells between washes. For single use and long term storage of more than 3 months, we recommend storing libraries at -80°C. For further information, please refer to the Library Stability Know-How document.
5. Priming and loading the PromethION Flow Cell
Material
- Sequencing Buffer (SB)
- Library Beads (LIB) (microesferas de carga de la biblioteca)
- Library Solution (LIS)
- Flow Cell Tether (FCT) (anclaje de celda de flujo)
- Flow Cell Flush (FCF)
Consumibles
- Celda de flujo PromethION
- Tubos de 1,5 ml Eppendorf DNA LoBind
Instrumental
- PromethION device
- PromethION Flow Cell Light Shield
- P1000 pipette and tips
- P200 pipette and tips
- Pipeta y puntas P20
IMPORTANTE
This kit is only compatible with R10.4.1 flow cells (FLO-PRO114M).
Descongelar los viales Sequencing Buffer (SB), Library Beads (LIB) o Library Solution (LIS), -si se requiere-, y un tubo de Flow Cell Flush (FCF) a temperatura ambiente. Agitar en vórtex, centrifugar y colocar en hielo.
To prepare the flow cell priming mix, combine Flow Cell Tether (FCT) and Flow Cell Flush (FCF), as directed below. Mix by vortexing at room temperature.
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.
Single-use tubes format: Add 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).
Bottle format: In a clean suitable tube for the number of flow cells, combine the following reagents:
| Reagent | Volume per flow cell |
|---|---|
| Flow Cell Flush (FCF) | 1,170 µl |
| Flow Cell Tether (FCT) | 30 µl |
| Total volume | 1,200 µl |
IMPORTANTE
Una vez sacadas de la nevera, esperar 20 minutos antes de insertar las celdas de flujo en el dispositivo y así darles tiempo a que estén a temperatura ambiente. En entornos húmedos se puede formar condensación. Inspeccione las clavijas doradas del conector, situadas en la parte superior e inferior de la celda de flujo, en busca de condensación y si la hubiera, límpiela con una toallita sin pelusa. Procure que la almohadilla térmica (color gris oscuro) esté enganchada en la parte posterior.
For PromethION 2 Solo, load the flow cell(s) as follows:
Place the flow cell flat on the metal plate.
Slide the flow cell into the docking port until the gold pins or green board cannot be seen.
For the PromethION 24/48, load the flow cell(s) into the docking ports:
- Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
- Press down firmly onto the flow cell and ensure the latch engages and clicks into place.
IMPORTANTE
Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.
Slide the inlet port cover clockwise to open.
IMPORTANTE
Tenga cuidado a la hora de extraer el tampón de la celda de flujo. No retire más de 20-30 μl y asegúrese de que el tampón cubra la matriz de poros en todo momento. La introducción de burbujas de aire en la matriz puede dañar los poros de manera irreversible.
After opening the inlet port, draw back a small volume to remove any air bubbles:
- Set a P1000 pipette tip to 200 µl.
- Insert the tip into the inlet port.
- Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.
Load 500 µl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes. During this time, prepare the library for loading using the next steps in the protocol.
Mezclar con la pipeta, minuciosamente, el contenido del vial Library Beads (LIB).
IMPORTANTE
Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.
En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
| Reagent | Volume per flow cell |
|---|---|
| Sequencing Buffer (SB) | 100 µl |
| Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS) | 68 µl |
| DNA library | 32 µl |
| Total | 200 µl |
Note: Library loading volume has been increased to improve array coverage.
Complete the flow cell priming by slowly loading 500 µl of the priming mix into the inlet port.
Mezclar la biblioteca pipeteando suavemente, justo antes de cargar.
Load 200 µl of library into the inlet port using a P1000 pipette.
Close the valve to seal the inlet port.
IMPORTANTE
Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.
Recomendamos colocar la pantalla protectora en la celda de flujo y dejarla puesta mientras la biblioteca esté cargada, incluyendo los lavados y pasos de recarga. Retirar la pantalla cuando se haya extraído la biblioteca de la celda de flujo.
If the light shield has been removed from the flow cell, install the light shield as follows:
- Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
- Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.
FIN DEL PROCESO
Close the PromethION lid when ready to start a sequencing run on MinKNOW.
Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.
6. Unloading the Tecan DreamPrep NGS worktable
Consumibles
- Low-lint scientific wipes (e.g. Kimberly-Clark™, cat # 7552 or equivalent)
- Double distilled water (ddH2O) (e.g. ThermoFisher, cat # 11983084)
- Freshly prepared ≥80% ethanol in nuclease-free or double distilled water, for cleaning
- ~10% Bleach (or equivalent): Thermo Scientific Alfa Aesar Sodium hypochlorite, 11-15% available chlorine, (e.g. ThermoFisher, cat # 15429019)
IMPORTANTE
Please ensure you have removed the plate containing your eluted sample libraries and stored it appropriately before unloading the rest of the worktable.
Empty the tip waste container.
Dispose of the used tips in an appropriate container.
Remove all the remaining plates from the worktable and hotel sites, and discard accordingly.
Remove the disposable 25 ml and 100 ml troughs from the trough mounting sites, and discard accordingly.
Note: Take care not to spill any residual liquid waste when removing from the worktable.
Remove the Sarstedt tubes from the POGO tube holder, and discard accordingly.
Clean the Bio-Rad™ Arched Auto-Sealing Lid:
- Wipe the lid with 10% bleach
- Thoroughly rinse the bleach off the lid using ≥80% ethanol or double distilled water (ddH2O) and lint-free wipes
- Allow the lid to air dry
Remove and/or restock the tip boxes on the worktable:
- Remove all Flexible Channel Arm (FCA) hanging tip trays from the FCA standard tip carriers, and discard accordingly.
- Remove all empty MultiChannel Arm (MCA) tip boxes from the worktable and discard accordingly.
- For partially used tip boxes, consider restacking the box with the appropriate tips for the next run.
Note: The MultiChannel Arm (MCA) tip boxes must be fully stacked. Failure to fully stack the MCA tip boxes can result in run error.
In cases where spillage has occurred during the automated library preparation, wipe the worktable surface using ≥80% ethanol.
FIN DEL PROCESO
Conclude all open dialogues on the TouchTools screen.
7. Data acquisition and basecalling
How to start sequencing
Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.
MinKNOW can be used and set up to sequence in multiple ways:
- On a computer either direcly or remotely connected to a sequencing device.
- Directly on a GridION or PromethION 24/48 sequencing device.
For more information on using MinKNOW on a sequencing device, please see the device user manuals:
To start a sequencing run on MinKNOW:
1. Navigate to the start page and click Start sequencing.
2. Fill in your experiment details, such as name and flow cell position and sample ID.
3. Select the sequencing kit used in the library preparation on the Kit page.
4. Configure the sequencing and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.
Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.
5. Click Start to initiate the sequencing run.
Identificación de bases duplex
La química del kit 14 ha mejorado la identificación de bases duplex, la cual requiere, tras realizar la identificación de bases simplex en MinKNOW, volver a identificar las bases en el programa Dorado con herramientas de identificación de bases duplex.
Para obtener información detallada sobre la configuración de un experimento de secuenciación, tanto para la identificación de bases simplex, como para la duplex, consultar la hoja informativa Kit 14 sequencing and duplex basecalling.
Nota: Mientras Dorado se esté ejecutando, recomendamos parar otros procesos de identificación de bases, para potenciar la memoria disponible en el programa. Esta acción se puede detener y reiniciar, cuando Dorado haya terminado, a través de la interfaz gráfica de MinKNOW.
Análisis de datos
Una vez la secuenciación ha finalizado, es posible reutilizar o devolver la celda de flujo, como se describe en la sección sobre Reutilización o retorno de celdas de flujo.
Tras secuenciar e identificar las bases, es posible analizar los datos. Si desea más información sobre las opciones de identificación de bases y de análisis posterior, consulte el documento Data Analysis.
En la sección Análisis posterior, se describen otras opciones para analizar los datos.
8. Reutilización y devolución de celdas de flujo
Material
- Flow Cell Wash Kit (EXP-WSH004) (kit de lavado de celda de flujo)
Si al terminar el experimento desea volver a usar la celda de flujo, siga las instrucciones del protocolo Flow Cell Wash Kit y guarde la celda de flujo lavada a entre 2 °C y 8 ⁰C.
El protocolo Flow Cell Wash Kit está disponible en la comunidad Nanopore.
CONSEJO
Una vez terminado el experimento, recomendamos lavar la celda de flujo cuanto antes. Si no es posible, se puede dejar en el dispositivo y lavar al día siguiente.
Otra posibilidad es seguir el procedimiento de devolución para lavar la celda de flujo y enviarla a Oxford Nanopore.
Aquí puede encontrar las instrucciones para devolver celdas de flujo.
IMPORTANTE
Ante cualquier duda o pregunta acerca del experimento de secuenciación, consulte la guía de resolución de problemas, Troubleshooting Guide, que se encuentra en la versión en línea de este protocolo.
9. Análisis
Análisis posterior a la identificación de bases
Existen varias opciones para completar el análisis de los datos de identificación de bases:
1. Procesos de trabajo en EPI2ME
Para realizar un análisis de datos exhaustivo, Oxford Nanopore Technologies ofrece una serie de tutoriales y procesos de trabajo de bioinformática, disponibles en EPI2ME Labs, situados en la sección EPI2ME de la comunidad Nanopore. La plataforma proporciona un espacio donde los procesos de trabajo que depositan en GitHub nuestros equipos de Investigación y Aplicaciones, se pueden exponer con textos descriptivos, código bioinformático funcional y datos de ejemplo.
2. Herramientas de análisis
El departamento de Investigación de Oxford Nanopore Technologies ha creado una serie de herramientas de análisis que están disponibles en el repositorio Oxford Nanopore de GitHub. Las herramientas están diseñadas para usuarios avanzados y contienen instrucciones sobre cómo instalar y ejecutar el programa. Estas herramientas están públicamente disponibles y cuentan con un mantenimiento mínimo.
3. Herramientas de análisis desarrolladas por la comunidad
Si en ninguno de los recursos anteriores se proporciona un método de análisis que responda a las necesidades de investigación requeridas, puede consultar la sección Bioinformatics del centro de recursos. Varios miembros de la comunidad Nanopore han desarrollado sus propias herramientas y cartera de productos en desarrollo para analizar los datos de la secuenciación por nanoporos. La mayoría de ellas está disponible en GitHub. Oxford Nanopore Technologies no desarrolla ni mantiene esas herramientas y no garantiza que sean compatibles con la última configuración de química/software.
10. Issues during automation of library preparation
Please contact your local Tecan Helpdesk and/or Nanopore FAS if you have any issues.
Your Tecan Helpdesk can be located via the following link: https://www.tecan.com/contact-us
11. Issues during the sequencing run
A continuación hay una lista de los problemas más frecuentes, con algunas posibles causas y soluciones propuestas.
También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.
Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.
Menos poros al inicio de la secuenciación que después de verificar la celda de flujo
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| MinKNOW presentó al inicio de la secuenciación un número de poros inferior al indicado durante la comprobación de la celda de flujo | Se introdujo una burbuja de aire en la matriz de nanoporos | Tras comprobar el número de poros presente en la celda de flujo, es imprescindible quitar las burbujas que haya cerca del puerto de cebado. Si no se quitan, pueden desplazarse a la matriz de nanoporos y dañar de manera irreversible los nanoporos expuestos al aire. En este vídeo se muestran algunas buenas prácticas para evitar que esto ocurra. |
| MinKNOW presentó al inicio de la secuenciación un número de poros inferior al indicado durante la comprobación de la celda de flujo | La celda de flujo no está colocada correctamente | Detener el ciclo de secuenciación, quitar la celda de flujo del dispositivo e insertarla de nuevo. Comprobar que está firmemente asentada en el dispositivo y que ha alcanzado la temperatura deseada. Si procede, probar con una posición diferente del dispositivo (GriION/PromethION). |
| MinKNOW presentó al inicio de la secuenciación un número de poros inferior al indicado durante la comprobación de la celda de flujo | La presencia de contaminantes en la biblioteca ha dañado o bloqueado los poros | El número de poros resultante tras la comprobación de la celda de flujo se realiza usando el control de calidad de las moléculas de ADN presentes en el tampón de almacenamiento de la celda de flujo. Al inicio de la secuenciación, se utiliza la misma biblioteca para estimar el número de poros activos. Por este motivo, se estima que puede haber una variabilidad del 10 % en el número de poros detectados. Tener un número de poros considerablemente inferior al inicio de la secuenciación puede deberse a la presencia de contaminantes en la biblioteca que hayan dañado las membranas o bloqueado los poros. Para mejorar la pureza del material de entrada tal vez sea necesario usar métodos de purificación o extracción de ADN/ARN alternativos. Los efectos de los contaminantes están descritos en la página Contaminants. Se recomienda, probar con un método de extracción alternativo que no provoque el arrastre de contaminantes. |
Error en el script de MinKNOW
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| MinKNOW muestra el mensaje "Error en el script" | Reiniciar el ordenador y reiniciar MinKNOW. Si el problema continúa, reúna los archivos de registro MinKNOW log files y contacte con el servicio de asistencia técnica. Si no dispone de otro dispositivo de secuenciación, recomendamos que guarde la celda de flujo con la biblioteca cargada a 4 °C y contacte con el servicio de asistencia técnica para recibir recomendaciones de almacenamiento adicionales. |
Pore occupancy below 40%
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Pore occupancy <40% | Not enough library was loaded on the flow cell | Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol" |
| Pore occupancy close to 0 | The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA | Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents. |
| Pore occupancy close to 0 | The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation | Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters. |
| Pore occupancy close to 0 | No tether on the flow cell | Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/FCF before priming. |
Longitud de lectura más corta de lo esperado
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| Longitud de lectura más corta de lo esperado | Fragmentación no deseada de la muestra de ADN | La longitud de lectura refleja la longitud del fragmento de la muestra de ADN. La muestra de ADN se puede fragmentar durante la extracción de la preparación de la biblioteca. 1. Consulte la sección de buenas prácticas de los métodos de extracción en la página Extraction Methods de la comunidad Nanopore. 2. Visualizar la distribución de la longitud de los fragmentos de las muestras de ADN en un gel de agarosa antes de proceder a la preparación de la biblioteca.  En la imagen superior, la muestra 1 contiene alto peso molecular, mientras que la muestra 2 se ha fragmentado.3. Durante la preparación de la biblioteca, evitar pipetear y agitar en vórtex cuando se mezclen los reactivos. Dar suaves golpes con el dedo o invertir el vial es suficiente. |
Gran proporción de poros no disponibles
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
Gran proporción de poros no disponibles (se muestran en azul oscuro en el panel de canales y en el gráfico de actividad de poros)  Conforme pasa el tiempo, el gráfico de actividad de poros de arriba muestra una proporción creciente de poros no disponibles. | Hay contaminantes presentes en la muestra | Algunos contaminantes se pueden eliminar de los poros mediante la función de desbloqueo incorporada en MinKNOW. Si funciona, el estado de los poros cambiará a "sequencing pores" (secuenciación de poros). Si la porción poros no disponibles se mantiene elevada o aumenta, pruebe una de las siguientes opciones: 1. Realizar un enjuague de nucleasa con el kit de lavado Flow Cell Wash Kit (EXP-WSH004) 2. Realizar varios ciclos de PCR para intentar diluir cualquier contaminante que pueda estar causando problemas. |
Gran proporción de poros inactivos
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| Gran proporción de poros inactivos/no disponibles (se muestran en azul claro en el panel de canales y en el gráfico de actividad de poros. Los poros o membranas están dañados de manera irreversible) | Se han introducido burbujas de aire en la celda de flujo | Las burbujas de aire introducidas durante el cebado de la celda y la carga de la biblioteca pueden dañar los poros de forma permanente. Para conocer las buenas prácticas de cebado y carga de la celda de flujo, ver el vídeo Priming and loading your flow cell |
| Gran proporción de poros inactivos/no disponibles | Ciertos compuestos copurificados con ADN | Compuestos conocidos, incluidos los polisacáridos, se asocian generalmente con el ADN genómico de las plantas. 1. Consulte la página Plant leaf DNA extraction method. 2. Limpiar usando el kit QIAGEN PowerClean Pro. 3. Realizar una amplificación del genoma completo con la muestra original de ADNg utilizando el kit QIAGEN REPLI-g. |
| Gran proporción de poros inactivos/no disponibles | Hay contaminantes presentes en la muestra | Los efectos de los contaminantes se muestran en la página Contaminants. Probar con un método de extracción alternativo que no provoque el arrastre de contaminantes. |
Reducción de la velocidad de secuenciación y del índice de calidad Qscore en una fase avanzada de la secuenciación
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| Reducción de la velocidad de secuenciación y el índice de calidad Qscore en una fase avanzada de la secuenciación | En la química del kit 9 (p. ej., SQK-LSK109), cuando la celda de flujo está sobrecargada con la biblioteca se observa un consumo rápido de combustible (consulte el protocolo correspondiente a su biblioteca de ADN para ver las recomendaciones) | Añadir más combustible a la celda de flujo, siguiendo las instrucciones en el protocolo de MinKNOW. En futuros experimentos, cargar cantidades menores de biblioteca en la celda de flujo. |
Fluctuación de la temperatura
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| Fluctuación de la temperatura | La celda de flujo ha perdido contacto con el dispositivo | Comprobar que una almohadilla térmica cubra la placa metálica de la parte posterior de la celda de flujo. Reinsertar la celda de flujo y presionar para asegurarse de que las clavijas del conector estén bien conectadas al dispositivo. Si el problema continúa, contacte con el servicio de asistencia técnica. |
Error al intentar alcanzar la temperatura deseada
| Observación | Posible causa | Comentarios y acciones recomendadas |
|---|---|---|
| MinKNOW muestra el mensaje "Error al intentar alcanzar la temperatura deseada" | El dispositivo ha sido colocado en un lugar a una temperatura ambiente inferior a la media o en un lugar con escasa ventilación (lo que provoca el sobrecalientamiento de las celdas de flujo). | MinKNOW tiene un tiempo predeterminado para que las celdas de flujo alcancen la temperatura fijada. Una vez transcurrido ese tiempo, aparece un mensaje de error, pero el experimento de secuenciación continua. Secuenciar a una temperatura incorrecta puede llevar a una disminución en el rendimiento y a generar un índice de calidad Qscore menor. Corrija la ubicación del dispositivo, procure que esté a temperatura ambiente y tenga buena ventilación; a continuación, reinicie el proceso en MinKNOW. Encontrará más información sobre el control de temperatura del MinION en este enlace. |
Guppy – no input .fast5 was found or basecalled
| Observation | Possible cause | Comments and actions |
|---|---|---|
| No input .fast5 was found or basecalled | input_path did not point to the .fast5 file location | The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH. |
| No input .fast5 was found or basecalled | The .fast5 files were in a subfolder at the input_path location | To allow Guppy to look into subfolders, add the --recursive flag to the command |
Guppy – no Pass or Fail folders were generated after basecalling
| Observation | Possible cause | Comments and actions |
|---|---|---|
| No Pass or Fail folders were generated after basecalling | The --qscore_filtering flag was not included in the command | The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders. |
Guppy – unusually slow processing on a GPU computer
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Unusually slow processing on a GPU computer | The --device flag wasn't included in the command | The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command. |
12. This protocol is for Research Use Only. Not for use in diagnostic procedures.
IMPORTANTE
Tecan Group Ltd. disclaimer
Tecan and DreamPrep are registered trademarks of Tecan Group Ltd., Männedorf, Switzerland. Flexible Channel Arm and MultiChannel Arm are trademarks of Tecan Group Ltd., Männedorf, Switzerland.
Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication; however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice.
For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative.
In addition, this brochure may contain reference to applications and products which are not available in all markets.
For more information, please check with your Tecan local sales representative.
Last updated: 10/4/2023
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