Ligation sequencing gDNA V14 - reduced representation methylation multiplex sequencing (RRMS) (SQK-NBD114.24)
- Home
- Documentation
- Ligation sequencing gDNA V14 - reduced representation methylation multiplex sequencing (RRMS) (SQK-NBD114.24)
PromethION: Protocol
Ligation sequencing gDNA V14 - reduced representation methylation multiplex sequencing (RRMS) (SQK-NBD114.24) V RRMS_9209_v114_revC_20Nov2024
For Research Use Only.
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Sample preparation
Library preparation
- 6. Reparación del ADN y preparación de los extremos (3)
- 7. Native barcode ligation
- 8. Adapter ligation and clean-up
- 9. Priming and loading the PromethION Flow Cell
- 10. Washing and reloading a PromethION Flow Cell
Sequencing and data analysis
- 11. Data acquisition and basecalling
- 12. Downstream analysis
- 13. Reutilización y devoluciones de las celdas de flujo (1)
Troubleshooting
Descripción general
For Research Use Only.
1. Overview of the protocol
IMPORTANTE
Adaptive sampling in Kit 14 chemistry
While using Kit 14 chemistry, this workflow has been optimised to enrich specific regions of interest (ROIs) with Adaptive sampling rather than duplex basecalling, ensuring highest output and the best sequencing results.
For more background information about designing an adaptive sampling experiment, please refer to the Adaptive sampling best practice document: Adaptive sampling best practice
Reduced representation methylation sequencing (RRMS)
Nanopore sequencing enables direct detection of methylated cytosines (e.g., at CpG sites), without the need for bisulphite conversion. CpG sites frequently occur in high density clusters called CpG islands (CGI) and most of vertebrate genes have their promoters embedded within CGIs.
Changes in methylation patterns within promoters is associated with changes in gene expression and disease states such as cancer: exploring methylation differences between tumour samples and normal samples can help to uncover mechanisms associated with tumour formation and development.
Adaptive sampling (AS) offers a fast, flexible and precise method to enrich for regions of interest (e.g. CGIs) by depleting off-target regions during sequencing itself with no requirement for upfront sample manipulation.
To read more about how the method works, and how it compares to other techniques for analysing methylation (e.g. EPIC arrays, bisulfite), please see our Introduction to Reduced-Representation Methylation Sequencing.
RRMS can be deployed on MinION Mk1b, GridION and PromethION P2S, P24 and P48 platforms.
When running on MinION/GridION, we recommend running a single sample per flow cell - using our Ligation sequencing gDNA V14 - reduced representation methylation sequencing (RRMS) (SQK-LSK114) protocol.
Alternatively, it is possible to multiplex up to 4 samples on a single PromethION flow cell, as outlined in this protocol.
Human sample sequencing
The RRMS protocol enables users to target 310 Mb of the human genome which are highly enriched for CpGs including all annotated CpG islands, shores, shelves and >90% of promoter regions (100% of promoter with more than 4 CpGs). As well as other rich CpG regions in the genome. The total number of CpG sites in the .bed file is 7.18 million.
For benchmarking purposes, we performed RRMS on five replicates of a metastatic melanoma cell line and its normal pair for a male individual (COLO829/COLO829_BL) and a triple negative breast cancer cell-line pair (HCC1395/HCC1935_BL). Each sample was run on a single MinION flow cell. RRMS resulted in high-confidence methylation calls (>10 overlapping reads) for 7.3-8.5 million CpGs per sample.
For comparison, we also performed Reduced Representation Bisulphite Sequencing (RRBS), which typically yields 1.7–2.5 high confidence calls per sample. More information on this comparison can be accessed in our RRMS performance document and poster.
Mouse sample sequencing
The RRMS protocol and a new .bed file have also been developed to target 308 Mb of the mouse genome, covering 100% of CpG island and promoter regions; as well as other rich CpG regions in the genome.
The performance of RRMS for mouse samples was characterised on replicates of a blastocyst-derived, embryonic stem cell line (ES-E14TG2a) and a leukemia cell-line (BALB/c AMuLV A.3R.1). A non-RRMS library was also run as a control. Each sample was run on a single MinION flow cell: RRMS resulted in high-confidence methylation calls (>10X reads per site) for 5.0–5.8 million CpGs per sample in the mouse genome, compared to ~400,000 CpGs in the control library.
Alternative vertebrate genomes could be sequenced using the RRMS protocol and a bespoke .bed file.
However, please note Oxford Nanopore Technologies has only validated this method using human and mouse samples.
Introduction to the DNA extraction and multiplex sequencing protocol for RRMS
This protocol describes how to carry out DNA extraction and reduced representation methylation sequencing (RRMS) for up to 4 samples on a single PromethION flow cell, using the Native Barcoding Kit (SQK-NBD114.24) and the Adaptive Sampling feature in MinKNOW.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Download the software for acquiring and analysing your data
- Ensure that you have the correct .bed file for Adaptive Sampling
- Check your flow cell to ensure it has enough pores for a good sequencing run
Sample preparation
- Extract your DNA using the QIAGEN Puregene Cell Kit.
- Fragment your DNA using the Covaris g-TUBE, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
Library preparation
The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.
Library preparation | Process | Time | Stop option |
---|---|---|---|
DNA repair and end-prep | Repair the fragmented DNA and prepare the DNA ends for barcode attachment | 35 minutes | 4°C overnight |
Native barcode ligation | Ligate the native barcodes to the DNA ends | 60 minutes | 4°C overnight |
Adapter ligation and clean-up | Attach the sequencing adapters to the barcoded DNA ends | 50 minutes | 4°C short-term storage or for repeated use, such as re-loading your flow cell -80°C for single-use, long-term storage. We strongly recommend sequencing your library as soon as it is adapted. |
Priming and loading the flow cell | Prime the flow cell and load the prepared library for sequencing | 5 minutes | |
Washing and reloading the flow cell (x2) | Pause your sequencing run. Wash your flow cell with nuclease to remove the previous library load and unblock pores. Prime the flow cell and reload the prepared library to continue sequencing | 60 minutes (x2) |
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. While configuring the run, turn on the Adaptive Sampling setting and import a pre-prepared .bed file with your regions of interest, along with a FASTA reference file.
- Sequence the sample for a total of 96 hours, with two flow cell washes when the available pore count drops to around 40% of the starting pore count (typically after ~24 hours and the second time after ~48 hours).
- Use Dorado to call modified bases, for more information please refer to the Dorado github page.
- Use the commands recommended at the end of this protocol to aggregate the modified bases and perform CpG island annotation.
IMPORTANTE
Compatibility of this protocol
This protocol should only be used in combination with:
- Native Barcoding Kit 24 V14 (SQK-NBD114.24)
- R10.4.1 PromethION Flow Cells (FLO-PRO114M)
- Flow Cell Wash Kit (EXP-WSH004)
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Native Barcoding Expansion V14 (EXP-NBA114)
- PromethION 24/48 device - PromethION IT requirements document
- PromethION 2 Solo device - PromethION 2 Solo IT requirements document
2. Equipment and consumables
Material
- 5 x 10^6 cells per sample, for 4 samples (FOR EXTRACTION)
- 2 µg of fragmented gDNA per sample, for 4 samples (FOR LIBRARY PREP)
- Native Barcoding Kit 24 V14 (SQK-NBD114.24)
- Flow Cell Wash Kit (EXP-WSH004) (kit de lavado de celda de flujo)
Consumibles
- Celda de flujo PromethION
- Puregene Cell Kit (QIAGEN, 158043)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- 1 x Phosphate-buffered saline (PBS)
- Isopropanol
- g-TUBE™ (Covaris, 520079)
- 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)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext® Quick Ligation Module (NEB, E6056)
- Etanol al 70 % recién preparado en agua sin nucleasas
- Etanol al 80 % recién preparado con agua sin nucleasas
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
- 15 ml Falcon tubes
- Tubos de 1,5 ml Eppendorf DNA LoBind
- Tubos de PCR de pared fina (0,2 ml)
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
Instrumental
- PromethION device
- PromethION Flow Cell Light Shield
- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Incubator or water bath set at 37°C and 50°C
- Inoculation loop or disposable tweezers for spooling DNA
- Eppendorf 5424 centrifuge (or equivalent)
- Mezclador Hula (mezclador giratorio suave)
- Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
- Microcentrífuga
- Mezclador vórtex
- Termociclador
- Wide-bore pipette tips
- Pipeta y puntas P1000
- Pipeta y puntas P200
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P10
- Pipeta y puntas P2
- Cubeta con hielo
- Temporizador
Equipo opcional
- Agilent Femto Pulse System (or equivalent for read length QC)
- Fluorímetro Qubit (o equivalente para el control de calidad)
IMPORTANTE
The above list of materials, consumables, and equipment is for the extraction method in the sample preparation section, as well as the library preparation section of the protocol. If you have pre-extracted sample(s), you will only require the materials for the library preparation section of this protocol.
For this protocol, the following inputs are required:
Input requirements per sample for the extraction method:
- 5 x 10^6 cells per sample
Input requirements per sample for the library preparation:
- 2 µg of g-tube fragmented gDNA
Cantidad de muestra inicial de ADN
Cómo realizar un control de calidad del ADN de la muestra inicial
Es importante que la muestra de ADN cumpla con los requisitos de cantidad y calidad. Usar demasiado ADN, poco o de mala calidad (p. ej., que esté muy fragmentado, que contenga ARN o contaminantes químicos), puede afectar a la preparación de la biblioteca.
Para realizar un control de calidad en la muestra de ADN, consulte el protocolo Input DNA/ RNA QC
Contaminantes químicos
Dependiendo de cómo se extraiga el ADN de la muestra cruda, ciertos contaminantes químicos pueden permanecer en el ADN purificado, lo cual afecta la eficacia de la preparación de la biblioteca y la calidad de la secuenciación. Encontrará más información sobre contaminantes en la página Contaminants de la comunidad Nanopore.
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.
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 |
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
The Native Adapter (NA) included in this kit and protocol is not interchangeable with other sequencing adapters.
Native Barcoding Kit 24 V14 (SQK-NBD114.24) contents
Note: We are in the process of reformatting the barcodes provided in this kit into a plate format. This will reduce plastic waste and will facilitate automated applications.
Plate format
Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
---|---|---|---|---|
DNA Control Sample | DCS | Yellow | 2 | 35 |
Native Adapter | NA | Green | 1 | 40 |
Sequencing Buffer | SB | Red | 1 | 700 |
Library Beads | LIB | Pink | 1 | 600 |
Library Solution | LIS | White cap, pink label | 1 | 600 |
Elution Buffer | EB | Black | 2 | 500 |
AMPure XP Beads | AXP | Clear cap, light teal label | 1 | 6,000 |
Long Fragment Buffer | LFB | Orange | 1 | 1,800 |
Short Fragment Buffer | SFB | Clear | 1 | 1,800 |
EDTA | EDTA | Blue | 1 | 700 |
Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
Flow Cell Tether | FCT | Purple | 1 | 200 |
Native Barcode plate | NB01-24 | - | 2 plates, 3 sets of barcodes per plate | 5 µl per well |
Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.
Vial format
Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
---|---|---|---|---|
Native Barcodes | NB01-24 | Clear | 24 (one per barcode) | 20 |
DNA Control Sample | DCS | Yellow | 2 | 35 |
Native Adapter | NA | Green | 1 | 40 |
Sequencing Buffer | SB | Red | 1 | 700 |
Library Beads | LIB | Pink | 1 | 600 |
Library Solution | LIS | White cap, pink label | 1 | 600 |
Elution Buffer | EB | Black | 2 | 500 |
AMPure XP Beads | AXP | Clear cap, light teal label | 1 | 6,000 |
Long Fragment Buffer | LFB | Orange | 1 | 1,800 |
Short Fragment Buffer | SFB | Clear | 1 | 1,800 |
EDTA | EDTA | Blue | 1 | 700 |
Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
Flow Cell Tether | FCT | Purple | 1 | 200 |
Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.
3. .bed file
Download the .bed file from the Adaptive Sampling catalogue.
The Adaptive Sampling catalogue provides a way for both the Oxford Nanopore team and Community members to share .bed files with genomic target regions used for Adaptive Sampling experiments. The .bed files along with a reference genome can be uploaded into MinKNOW.
For human genome RRMS experiments, download the Human reduced representation methylation sequencing (RRMS) file.
For mouse genome RRMS experiments, download the Mouse reduced representation methylation sequencing (RRMS) file.
(Optional): For alternative vertebrate genomes, please use a bespoke .bed file for the desired organism.
4. DNA extraction
Material
- 5 x 10^6 cells
Consumibles
- Puregene Cell Kit (QIAGEN, 158043)
- Etanol al 70 % recién preparado en agua sin nucleasas
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- 1 x Phosphate-buffered saline (PBS)
- Isopropanol
- Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- 15 ml Falcon tubes
- 1.5 ml Eppendorf DNA LoBind tubes
Instrumental
- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Incubator or water bath set at 37°C and 50°C
- Mezclador vórtex
- Inoculation loop or disposable tweezers for spooling DNA
- Wide-bore pipette tips
- Pipeta y puntas P1000
- Pipeta y puntas P200
- Pipeta y puntas P100
- Pipeta y puntas P20
- Fluorímetro Qubit (o equivalente para el control de calidad)
Extraction from cultured cell lines:
Extract DNA from your sample(s) using one of our recommended extraction protocols.
For the benchmarking of this method, the Oxford Nanopore team extracted DNA from ~5 million cells using the protocol: Human cell line DNA – QIAGEN Puregene Cell Kit. The steps for this method are outlined below.
Note: this method is also suitable for mouse cell line DNA.
We also offer multiple mammalian sample extraction protocols, which you can use for other sample types.
Harvest and pellet 5 x 10^6 cells by centrifugation at 300 x g for 3 minutes. If any liquid remains associated with the pellet, spin down the cells again and aspirate the remaining supernatant.
Add 200 µl of 1x PBS to the pelleted cells and centrifuge at 300 x g for 3 minutes. Aspirate and discard the supernatant.
Add 2 ml of Cell Lysis Solution to the washed cell pellet. Using a wide-bore pipette tip, resuspend the cells and transfer them to a 15 ml Falcon tube. If clumps of cells remain, gently invert the tube.
Incubate the sample at 37°C for 30 minutes.
Add 700 µl of the Protein Precipitation Solution to the lysed cells and mix by vortexing for three pulses of 5 seconds.
Centrifuge the sample at 2000 x g for 5 minutes.
Transfer the supernatant to a new tube and add 2.5 ml of room temperature isopropanol. Discard the pellet.
Mix by gently inverting the tube 50 times.
Spool the DNA using an inoculation loop or disposable tweezers.
Dip the spooled DNA in an Eppendorf tube containing 70% cold ethanol.
Remove the inoculation loop or tweezers with the spooled DNA from the ethanol tube, and allow it to air-dry for a few seconds.
Dip the DNA in a 1.5 ml Eppendorf DNA LoBind tube containing 250 µl TE (1 mM EDTA, pH 8.0) and allow the DNA to gently dislodge from the loop/tweezers.
Incubate the DNA pellet for 2 hours at 50°C, occasionally mixing the tube contents by gentle inversion.
Note: The pellet may take some time to dissolve, so ensure the solution is homogenous before quantifying.
Quantify 1 µl of each eluted sample using a Qubit fluorometer.
FIN DEL PROCESO
Take forward 2 µg of extracted gDNA, for each sample, into the fragmentation of extracted DNA stage of the protcol.
5. DNA fragmentation
Material
- 2 µg of extracted gDNA (from previous step)
Consumibles
- g-TUBE™ (Covaris, 520079)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
Instrumental
- Eppendorf 5424 centrifuge (or equivalent)
- Pipeta y puntas P1000
- Pipeta y puntas P200
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P2
- Fluorímetro Qubit (o equivalente para el control de calidad)
Equipo opcional
- Agilent Femto Pulse System (or equivalent for read length QC)
Fragmentation of extracted DNA using Covaris g-Tube:
To prepare fragmented gDNA for the library prep protocol, mechanical fragmentation is performed using a g-TUBE (Covaris) to shear DNA to a fragment length of approximately 6kb.
Prepare the DNA in TE buffer:
- Ensure you have 2 µg of extracted gDNA from the sample extraction, and transfer this into a 1.5 ml Eppendorf tube.
- Adjust the volume to 50 μl with TE buffer.
- Mix thoroughly by pipetting up and down.
- Spin down briefly in a microfuge.
Load the 50 µl of the sample into the top of the g-TUBE. Screw the cap firmly and centrifuge at 11,000 rpm (~11,300 RCF) for 30 seconds.
After centrifugation, spin the tube again at 11,000 rpm (~11,300 RCF) for 10 seconds to ensure complete passage of all gDNA through the constriction.
Visually inspect to confirm the entire sample has passed through the upper chamber to the lower chamber of the g-TUBE.
Invert the g-TUBE and spin it again at the same speed and duration as above: 11,000rpm (~11,300 RCF) for 30 seconds.
Repeat the centrifugation at 11,000 rpm (~11,300 RCF) for 10 seconds to ensure thorough passage of all gDNA through the constriction.
Unscrew the tube body, leaving the screw-cap containing the sample. Retrieve the sample from the g-TUBE screw-cap and transfer it into a clean 1.5 ml Eppendof tube.
Quantify 1 µl of the fragmented gDNA using the Qubit dsDNA Broad Range Assay Kit.
Sample concentration after g-TUBE shearing, is expected to be within the range of 25–35 ng/µl.
MEDIDA OPCIONAL
The fragmented gDNA should also be assessed using Femto-Pulse (Agilent) to evaluate the size and quality of the DNA.
Example DNA fragment distribution after g-tube fragmentation, analysed using an Agilent 165 kb Femto-Pulse Assay. Note the single prominent peak ~6 kb.
FIN DEL PROCESO
Take forward 2 µg of fragmented gDNA in 48 µl, for each sample, into the library preparation section of the protcol.
6. Reparación del ADN y preparación de los extremos (3)
Material
- 2 µg of fragmented gDNA in 48 µl per sample (4 samples)
- AMPure XP Beads (AXP) (microesferas magnéticas)
Consumibles
- NEBNext FFPE DNA Repair Mix (NEB M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB E7546)
- Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
- Etanol al 80 % recién preparado con agua sin nucleasas
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- Tubos de PCR de pared fina (0,2 ml)
- Tubos de 1,5 ml Eppendorf DNA LoBind
Instrumental
- Pipeta y puntas P1000
- Pipeta y puntas P100
- Pipeta y puntas P10
- Microcentrífuga
- Termociclador
- Mezclador Hula (mezclador giratorio suave)
- Gradilla magnética
- Cubeta con hielo
Equipo opcional
- Fluorímetro Qubit (o equivalente para el control de calidad)
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.
Preparación del ADN en agua sin nucleasas: (3)
Transferir 1 μg (o 100-200 fmol) de muestra de ADN a un tubo de 1,5 ml Eppendorf DNA LoBind.
Ajustar el volumen a un total de 47 μl con agua sin nucleasas.
Mezclar minuciosamente con la pipeta o golpear el tubo suavemente con el índice.
Centrifugar brevemente
En un tubo de PCR de pared fina (0,2 ml), mezclar lo siguiente: (3)
Entre cada adición, mezclar con la pipeta de 10 a 20 veces.
Reactivo | Volumen |
---|---|
ADN del paso anterior | 47 µl |
(opcional) DNA Control Sample (DCS) | 1 µl |
NEBNext FFPE DNA Repair Buffer | 3,5 µl |
NEBNext FFPE DNA Repair Mix | 2 µl |
Ultra II End-prep Reaction Buffer | 3,5 µl |
Ultra II End-prep Enzyme Mix | 3 µl |
Total | 60 µl |
Mezclar pipeteando con suavidad y centrifugar brevemente la reacción para asegurarse de que se mezcla completamente.
Using a thermal cycler with a heated lid, incubate the reaction at 20°C for 5 minutes, 60°C for 5 minutes and hold at 4°C.
Remove the reaction from the thermal cycler and place the tube on ice.
Resuspender las microesferas magnéticas AMPure XP Beads (AXP) agitándolas en vórtex.
Add a 1x volume (60 µl) of resuspended the AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.
Incubar en el mezclador Hula (o mezclador giratorio suave) durante 5 minutos a temperatura ambiente.
Prepare 3 ml of fresh 80% ethanol in nuclease-free water.
Note: Ensure you prepare sufficient 80% ethanol for your 4 samples.
Centrifugar la muestra y precipitar en un imán hasta que el sobrenadante se vuelva claro e incoloro. Dejar el tubo en el imán y retirar el sobrenadante con una pipeta.
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
Repetir el paso anterior.
Centrifugar y colocar el tubo de nuevo en el imán. Retirar con una pipeta cualquier residuo de etanol. Dejar secar el agregado durante 30 s aproximadamente, sin dejar que se agriete.
Quitar el tubo de la gradilla magnética y resuspender el agregado en 61 µl de agua sin nucleasas. Incubar durante 2 minutos a temperatura ambiente. (2)
Precipitar las microesferas en un imán, durante al menos 1 minuto, hasta que el eluido se vuelva claro e incoloro.
Extraer 61 µl de eluido y guardar en un tubo Eppendorf DNA Lobind de 1,5 ml. (2)
Note: Ensure your samples are processed separately. At this stage they are not yet barcoded.
CHECKPOINT
Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit. (3)
Note: We recommend performing multiple (triplicate) qubit readings of each of the 4 samples to quantify them more accurately. This will be essential for further normalizing of each sample before barcoding.
You should expect to recover 1000–1600 ng per sample after end-prep.
Using your quantification results, normalise your samples to the sample with the lowest yield.
- Take forward 15 µl of your lowest performing sample into a clean 0.2 ml thin-walled PCR tube.
- Take forward an equivalent mass of each of the other samples into separate clean 0.2 ml thin-walled PCR tubes.
- Adjust the volume of each of the samples 15 µl using nuclease-free water.
FIN DEL PROCESO
Una vez el ADN está reparado y con los extremos preparados, se puede proceder a la ligación del adaptador. En este punto, también se puede guardar la muestra a 4 ⁰C hasta el día siguiente. (3)
7. Native barcode ligation
Material
- End-prepped DNA in 15 µl from previous step (4 samples, normalised to the lowest yield sample)
- Native Barcodes (NB01-24)
- AMPure XP Beads (AXP) (microesferas magnéticas)
- EDTA (EDTA)
- Short Fragment Buffer (SFB) (tampón para fragmentos cortos)
Consumibles
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- Etanol al 80 % recién preparado con agua sin nucleasas
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- OR 0.2 ml thin-walled PCR tubes
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
Instrumental
- Gradilla magnética
- Mezclador vórtex
- Mezclador Hula (mezclador giratorio suave)
- Microfuge
- Termociclador
- Cubeta con hielo
- Multichannel pipette and tips
- Pipeta y puntas P1000
- P200 pipette and tips
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P10
- P2 pipette and tips
- Fluorímetro Qubit (o equivalente para el control de calidad)
Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:
Thaw the reagents at room temperature.
Spin down the reagent tubes for 5 seconds.
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Thaw the EDTA at room temperature and mix by vortexing. Then spin down and place on ice.
Thaw the Native Barcodes (NB01-24) at room temperature. Briefly spin down, individually mix the barcodes required for your number of samples by pipetting, and place them on ice.
Select a unique barcode for each sample to be run together on the same flow cell. 4 samples should be barcoded and combined in one experiment.
Please note: Only use one barcode per sample.
In the 0.2 ml PCR-tubes containing your normalised sample inputs, add the reagents in the following order for each sample:
Reagent | Volume |
---|---|
End-prepped DNA | 15 µl |
Native Barcode (NB01-24) | 5 µl |
Blunt/TA Ligase Master Mix | 20 µl |
Total | 40 µl |
Mezclar pipeteando con suavidad y centrifugar brevemente la reacción para asegurarse de que se mezcla completamente.
Incubate for 20 minutes at room temperature.
Add the following volume of EDTA to each well and mix thoroughly by pipetting and spin down briefly.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
EDTA cap colour | Volume per well |
---|---|
For clear cap EDTA | 2 µl |
For blue cap EDTA | 4 µl |
CONSEJO
EDTA is added at this step to stop the reaction.
Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
. | For 4 samples |
---|---|
Total volume for preps using clear cap EDTA | 168 µl |
Total volume for preps using blue cap EDTA | 176 µl |
CONSEJO
We recommend checking the base of your tubes/plate are all the same volume before pooling and after to ensure all the liquid has been taken forward.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 0.65X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting.
Note: Ensure you follow the instructions for the cap colour of your EDTA tube.
. | For 4 samples |
---|---|
Volume of AXP for preps using clear cap EDTA | 110 µl |
Volume of AXP for preps using blue cap EDTA | 115 µl |
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
IMPORTANTE
The next clean-up step uses Short Fragment Buffer (SFB) rather than 80% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.
Spin down the sample and pellet on a magnet for 5 minutes. Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.
Wash the beads by adding 500 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Repetir el paso anterior.
Spin down and place the tube back on the magnetic rack. Pipette off any residual Short Fragment Buffer (SFB). Allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 32 µl nuclease-free water by gently flicking.
Incubate for 15 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
Remove and retain 32 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
CHECKPOINT
Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit. (2)
Note: You should expect to recover between 2200–3200 ng following barcode ligation.
FIN DEL PROCESO
Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, you may store the sample at 4°C overnight.
8. Adapter ligation and clean-up
Material
- Long Fragment Buffer (LFB) (tampón para fragmentos largos)
- Elution Buffer (EB)
- Native Adapter (NA)
- AMPure XP Beads (AXP) (microesferas magnéticas)
Consumibles
- NEBNext® Quick Ligation Module (NEB, E6056)
- Tubos de 1,5 ml Eppendorf DNA LoBind
- Tubos de ensayo Qubit™ (Invitrogen Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
Instrumental
- Microcentrífuga
- Gradilla magnética
- Mezclador vórtex
- Mezclador Hula (mezclador giratorio suave)
- Termociclador
- Pipeta y puntas P1000
- Pipeta y puntas P200
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P10
- Ice bucket with ice
- Fluorímetro Qubit (o equivalente para el control de calidad)
IMPORTANTE
The Native Adapter (NA) used in this kit and protocol is not interchangeable with other sequencing adapters.
Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice:
Thaw the reagents at room temperature.
Spin down the reagent tubes for 5 seconds.
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes. Note: Do NOT vortex the Quick T4 DNA Ligase.
The NEBNext Quick Ligation Reaction Buffer (5x) may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.
IMPORTANTE
Do not vortex the Quick T4 DNA Ligase.
Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.
Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
Thaw the Long Fragment Buffer (LFB) at room temperature and mix by vortexing. Then spin down and place on ice.
In a 1.5 ml Eppendorf LoBind tube, mix in the following order:
Between each addition, pipette mix 10 - 20 times.
Reagent | Volume |
---|---|
Pooled barcoded sample | 30 µl |
Native Adapter (NA) | 5 µl |
NEBNext Quick Ligation Reaction Buffer (5X) | 10 µl |
Quick T4 DNA Ligase | 5 µl |
Total | 50 µl |
Mezclar pipeteando con suavidad y centrifugar brevemente la reacción para asegurarse de que se mezcla completamente.
Incubate the reaction for 20 minutes at room temperature.
IMPORTANTE
The next clean-up step uses Long Fragment Buffer (LFB) rather than 80% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 25 µl (0.5x) of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
Wash the beads by adding 250 μl Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet for at least 5 minutes. Remove the supernatant using a pipette and discard.
Note: Take care when removing the supernatant, the viscosity of the buffer can contribute to loss of beads from the pellet.
Repetir el paso anterior.
Centrifugar y colocar el tubo de nuevo en el imán. Retirar con una pipeta cualquier residuo de sobrenadante. Dejar secar el agregado durante 30 s aproximadamente, sin dejar que se agriete.
Remove the tube from the magnetic rack and resuspend the pellet in 97 µl of Elution Buffer (EB).
Spin down and incubate for 20 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Precipitar las microesferas en un imán, durante al menos 1 minuto, hasta que el eluido se vuelva claro e incoloro.
Remove and retain 97 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
CHECKPOINT
Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit. (2)
Note: You should expect to recover 1000–1200 ng after adapter ligation and clean-up in a volume of 96 µl.
FIN DEL PROCESO
La biblioteca preparada se usará para cargar la celda de flujo. Conservar la biblioteca en hielo o a 4 °C hasta el momento de cargar.
CONSEJO
Recomendaciones de guardado de la biblioteca
Se recomienda guardar las bibliotecas en tubos Eppendorf DNA LoBind a 4 ⁰C, durante periodos de tiempo cortos o en caso de uso repetido, por ejemplo, para recargar celdas de flujo entre lavados. Para uso individual y para conservar a largo plazo por periodos de más de 3 meses, se recomienda guardar las bibliotecas a -80 ⁰C en tubos Eppendorf DNA LoBind.
9. Priming and loading the PromethION Flow Cell
Material
- Sequencing Buffer (SB)
- Library Beads (LIB) (microesferas de carga de la biblioteca)
- 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 2 Solo device
- Dispositivo PromethION 24/48
- 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. (1)
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 | 68 µl |
DNA library | 32 µl |
Total | 200 µl |
Note: The prepared library is used for loading into the flow cell. Store the library on ice or at 4°C until ready to load.
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.
For instructions on setting up your sequencing run please visit the Data acquisition and basecalling section of this protocol.
Reminder: For this protocol, we recommend washing and reloading your flow cell with fresh library to maintain high data acquisition after ~24 hours of sequencing.
Follow the instructions in the Washing and reloading a PromethION Flow Cell section of this protocol.
10. Washing and reloading a PromethION Flow Cell
Material
- Adapter ligated DNA library (from previous step)
- Flow Cell Wash Kit (EXP-WSH004) (kit de lavado de celda de flujo)
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
Consumibles
- Tubos de 1,5 ml Eppendorf DNA LoBind
Instrumental
- Pipeta y puntas P1000
- Pipeta y puntas P20
- Cubeta con hielo
- Vortex mixer
We recommend washing and reloading the flow cell after ~24 hours of sequencing.
For this method, the flow cell is washed after ~24 hours of sequencing to restore pores to ensure efficient data acquisition. After an additional 24 hours of sequencing, the flow cell is washed and reloaded a second time. For this reason, enough library was generated for 3 flow cell loads in the adapter ligation step of the protocol.
- This washing procedure aims to remove most of the initial library and unblock the pores to prepare the flow cell for the loading of a subsequent library.
- Data acquisition in MinKNOW should be paused during the wash procedure and library loading.
- After the flow cell has been washed, the next library can be loaded.
You can navigate to the Pore Activity or the Pore Scan Results plot to see pore availability.
Below you can find example data for pore states observed on a flow cell before and after wash steps are performed. Additionally, you can observe an example for the cummulative sequencing data output, including the wash and reload steps. The red asterisks indicate the flow cell wash and reloads.
Figure 1. Channel state for a 96 hour run. The flow cell washes are incorporated into the method to restore blocked pores, to allow continuous data acquisition. Red asterisks denote when a flush was performed.
Figure 2. Cumulative sequencing data output, over a 96 hour run. Red asterisks denote when a flush was performed.
CONSEJO
We recommend keeping the light shield on the flow cell during washing if a second library will be loaded straight away.
If the flow cell is to be washed and stored, the light shield can be removed.
Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
Thaw one tube of Wash Diluent (DIL) at room temperature.
Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.
In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the following Flow Cell Wash Mix:
Reagent | Volume per flow cell |
---|---|
Wash Mix (WMX) | 2 μl |
Wash Diluent (DIL) | 398 μl |
Total | 400 μl |
Mix well by pipetting, and place on ice. Do not vortex the tube.
Pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
IMPORTANTE
It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Remove waste buffer, as follows:
- Close the inlet port.
- Insert a P1000 pipette into a waste port and remove the waste buffer.
Note: As both the inlet port is closed, no fluid should leave the sensor array area.
Slide the inlet port cover clockwise to open the inlet port.
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, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
- Set a P1000 pipette to 200 µl
- Insert the tip into the inlet port
- Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.
Slowly load 200 µl of the prepared flow cell wash mix into the inlet port, as follows:
- Using a P1000 pipette, take 200 µl of the flow cell wash mix
- Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
- Set a timer for a 5 minute incubation.
Once the 5 minute incubation time is complete, carefully load the remaining 200 µl of the prepared flow cell wash mix into the inlet port, as follows:
- Using a P1000 pipette, take 200 µl of the flow cell wash mix
- Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Close the inlet port and wait for 1 hour.
IMPORTANTE
It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Remove the waste buffer, as follows:
- Ensure the inlet port is closed.
- Insert a P1000 pipette into a waste port and remove the waste buffer
Note: As the inlet port is closed, no fluid should leave the sensor array area.
IMPORTANTE
The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.
Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature, before mixing by vortexing. Then spin down before storing on ice.
Prepare the flow cell priming mix in a suitable tube for the number of flow cells to flush. Once combined, mix well by briefly vortexing.
Reagents | Volume per flow cell |
---|---|
Flow Cell Flush (FCF) | 1,170 µl |
Flow Cell Tether (FCT) | 30 µl |
Total volume | 1,200 µl |
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.
Slowly load 500 µl of the priming mix into the inlet port, as follows:
- Using a P1000 pipette, take 500 µl of the priming mix
- Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
IMPORTANTE
It is vital to wait five minutes between the priming mix flushes to ensure effective removal of the nuclease.
Close the inlet port and 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.
IMPORTANTE
It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Remove the waste buffer, as follows:
- Ensure the inlet port is closed.
- Insert a P1000 pipette into a waste port and remove the waste buffer
Note: As the inlet port is closed, no fluid should leave the sensor array area.
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.
Slowly load 500 µl of the priming mix into the inlet port, as follows:
- Using a P1000 pipette, take 500 µl of the priming mix
- Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
IMPORTANTE
It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Remove waste buffer, as follows:
- Close the inlet port.
- Insert a P1000 pipette into a waste port and remove the waste buffer.
Note: As both the inlet port is closed, no fluid should leave the sensor array area.
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.
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.
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.
FIN DEL PROCESO
Perform the "Washing and reloading a PromethION flow cell" step twice for a total of three library loads (initial library load + two wash and reloads) to maximise data acquisition.
- The first wash and reload should be performed at ~24 hours into sequencing.
- The second wash and reload should be performed at ~48 hours into sequencing.
11. Data acquisition and basecalling
Aspectos generales del análisis de datos de nanoporos
Para obtener una descripción completa del análisis de datos de nanoporos, que incluya distintas posibilidades para el análisis de identificación y postidentificicación de bases, consultar el documento Data Analysis.
How to start sequencing
The sequencing device control and data acquisition are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer. Further instructions for setting up your sequencing run can be found in the MinKNOW protocol.
Sequencing settings for the reduced representation methylation multiplex sequencing protocol:
Select the Native Barcoding Sequencing Kit 24 (SQK-NBD114.24) in kit selection.
Turn basecalling OFF (this will automatically turn off barcoding).
Note: Basecalling and barcoding will be carried out post-sequencing in the downstream analysis section of the protocol.Turn Adaptive Sampling ON, and select Enrich.
Input the human reference file for alignment and the .bed file for enumerating regions (check online catalogue for the human RRMS .bed file).Set the run duration for a minimum of 96 hours.
Set up your desired output parameters.
To ensure the downstream analysis functions correctly, we recommend keeping the default options of the output file format (.POD5).Click “Start” begin the sequencing run.
12. Downstream analysis
IMPORTANTE
Software versions
See below the software versions used in this guide. Please note, newer versions of the software may not be compatible with commands shown in this guide.
Software | Version |
---|---|
dorado | v0.7.3 |
modkit | v0.2.8 |
wf-human-variation | v2.3.0 |
mosdepth | v0.3.8 |
Basecalling and demux:
Basecalling:
Dorado stand-alone is used for basecalling using the dorado basecaller. Open a terminal and enter the following commands:
dorado basecaller hac,5mCG_5hmCG --kit-name SQK-NBD114-24 \
--secondary “no” -Y \
--reference {reference_fasta} {input_pod5_folder} \
| samtools view -e '[qs] >= {qscore_filter}' \
--output {out_pass_bam} \
--unoutput {out_fail_bam}
Notes:
- We recommend using the high accuracy model (hac) for RRMS sequencing runs. However, if using the super accurate model (sup), ensure you are utilizing the correct model in the above command.
- Alignment can be performed while basecalling by providing a reference FASTA file, the recommended human reference file can be downloaded here.
- Secondary alignments are discarded by using “--secondary no” and -Y option is enabled, to allow soft-clipping supplementary alignments.
- By providing the option “--kit-name SQK-NBD114-24” dorado will also classify reads into the different barcodes present by adding a tag to the generated BAM file.
- We recommend setting the qscore filter to 10.
- Please note, GPU compute is needed to perform basecalling with dorado, more information on how to run dorado can be found in the github repository.
Demux
Dorado demux is used to sort reads per barcode using the following command:
dorado demux --no-classify --sort-bam --output-dir <out_folder> {out_pass_bam}
Notes:
- This step will generate sorted BAM files for each of the possible barcodes for kit used (e.g. SQK-NBD114-24).
- Trimming of adapters and barcodes will happen by default in dorado. Once barcodes are trimmed reads can not be demuxed again.
- For more information check the dorado documentation.
Coverage analysis:
RRMS target bed file can be downloaded from the AS catalogue available here.
Mosdepth is used to check coverage on target regions for the barcodes of interest:
mosdepth -x -t 8 -n -b {target_bed} {out_prefix} {input_pass_bam}
Modification calling:
Human variation pipeline is used to aggregate modifications per genomic positions using modkit.
The workflow is available in the following repository: wf-human-variation github.
The documentation can be found in the following space: wf-human-variation EPI2ME page
Modification calling:
For most RRMS runs we recommend running the following command:
nextflow run https://github.com/epi2me-labs/wf-human-variation \
-profile singularity \
--mod \
--bam <bam> \
--bed RRMS_human_hg38.bed \
--ref GCA_000001405.15_GRCh38_no_alt_analysis_set.fasta \
--sample_name <sample> --out_dir <output_dir>
(Optional) For haplotype-specific methylation:
If haplotype-specific methylation is required, you can provide options “--snp –phased“ to aggregate modifications identified on each of the haplotypes (i.e. one bedmethyl file for each of the haplotypes will be generated):
nextflow run https://github.com/epi2me-labs/wf-human-variation \
-profile singularity \
--mod --snp --phased \
--bam <bam> \
--bed RRMS_human_hg38.bed \
--ref GCA_000001405.15_GRCh38_no_alt_analysis_set.fasta \
--sample_name <sample> --out_dir <output_dir>
Note: For this specific analysis, a sample coverage of >30X is recommended.
Differentially methylated regions detection:
For detection of differentially methylated regions across different samples “modkit dmr” can be used.
For more information check the modkit documentation available here.
Visualisation:
The BAM file(s) generated by dorado contains canonical bases as well as per-read modifications stored in MM and ML BAM tags. To visualise the per-read modification calls, IGV can be used to load the BAM file and set "colour reads as" to “base modification 2-color (all)”.
If phasing was performed using wf-human-variation pipeline, the haplotagged BAM file can be uploaded in IGV and alignments can be grouped by haplotype using the IGV option “group by” and selecting “phase”.
Per-position methylation frequencies can also be visualised in IGV by using BIGWIG format. For this, modkit is used to generate BEDGRAPH files using the following command:
modkit pileup --cpg --combine-strands --bedgraph \
--threads 10 --prefix {out_prefix} \
--ref {reference_fasta} \
{out_folder} {input_pass_bam}
Please note, a different bedgraph file will be created for each of the modifications present, in this case 5mC and 5hmC.
Next, bedGraphToBigWig is used to generate bigwig files which can be uploaded together with your BAM file in IGV:
bedtools sort -i {out_folder}/{prefix}_m_CG0_combined.bedgraph | cut -f 1-4 > {out_folder}/{prefix}_m_CG0_combined_sort.bedgraph
bedGraphToBigWig {out_folder}/{prefix}_m_CG0_combined_sort.bedgraph {reference_chrSize} {out_mod_bed_agg_filt_bigwig}
Benchmarking results
For information about benchmarking the performance of RRMS for human samples, please see our RRMS performance document
13. Reutilización y devoluciones de las celdas de flujo (1)
We do not recommend washing and reusing your flow cells for this method.
Due to the extended sequencing time, and the multiple flow cell washes and library reloads, we do not recommend re-using the flow cells used in this method.
Re-using these flow cells for subsequent sequencing experiments may result in insufficient data generation for analysis.
Otra posibilidad es seguir el procedimiento de devolución para lavar la celda de flujo y enviarla a Oxford Nanopore. (1)
Aquí puede encontrar las instrucciones para devolver celdas de flujo.
Nota: Antes de proceder a su devolución, las celdas de flujo deben lavarse con agua desionizada.
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.
14. Problemas durante la extracción de ADN/ARN y la preparación de bibliotecas
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.
Baja calidad de la muestra
Observación | Posible causa | Comentarios y acciones recomendadas |
---|---|---|
Baja pureza del ADN (la lectura del Nanodrop para ADN OD 260/280 es <1,8 y OD 260/230 es <2,0-2,2) | El método de extracción de ADN no proporciona la pureza necesaria | Los efectos de los contaminantes se muestran en la página Contaminants. Pruebe con un método de extracción alternativo que no provoque el arrastre de contaminantes. Considere realizar un paso adicional de limpieza SPRI. |
Baja integridad del ARN (número de integridad del ARN <9,5 RIN o la banda ARNr se muestra como una mancha en el gel). | El ARN se degradó durante la extracción | Probar un método de extracción de ARN diferente. Encontrará más información sobre RIN en la página RNA Integrity Number. Asimismo, dispone de información adicional en la página DNA/RNA Handling. |
El ARN tiene una longitud de fragmento más corta de lo esperado | El ARN se degradó durante la extracción | Probar un método de extracción de ARN diferente. Encontrará más información sobre RIN en la página RNA Integrity Number. Asimismo, dispone de información adicional en la página DNA/RNA Handling. Cuando se trabaje con ARN, recomendamos que el espacio de trabajo y el instrumental de laboratorio estén libres de ribonucleasas. |
Escasa recuperación de ADN tras la limpieza con microesferas magnéticas AMPure
Observación | Posible causa | Comentarios y acciones recomendadas |
---|---|---|
Escasa recuperación | Pérdida de ADN debido a una proporción de microesferas magnéticas AMPure por muestra inferior a lo previsto. | 1. Las microesferas magnéticas AMPure precipitan con rapidez; antes de añadirlas a la muestra hay que asegurarse de que estén bien resuspendidas. 2. Si la proporción de microesferas por muestra es inferior a 0.4:1, los fragmentos de ADN, sean del tamaño que sean, se perderán durante la limpieza. |
Escasa recuperación | Los fragmentos de ADN son más cortos de lo esperado | Cuanto menor sea la proporción de microesferas magnéticas AMPure por muestra, más rigurosa será la selección de fragmentos largos frente a los cortos. Determinar siempre la longitud de la muestra de ADN en un gel de agarosa u otros métodos de electroforesis en gel, y, a continuación, calcular la cantidad adecuada de microesferas magnéticas que se debe utilizar. |
Escasa recuperación tras la preparación de extremos | El paso de lavado utilizó etanol a <70 % | Cuando se utilice etanol a <70 %, el ADN se eluirá de las microesferas magnéticas. Asegúrese de utilizar el porcentaje correcto. |
15. 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. |