Human variation sequencing from saliva samples using SQK-LSK114

Oxford Nanopore Technologies
Document type icon

PromethION: Protocol

Human variation sequencing from saliva samples using SQK-LSK114 V HVSA_9216_v114_revA_22Jan2025

End-to-end method outlining sample extraction, library preparation, sequencing and data analysis. This protocol:

  • Uses genomic DNA extracted from human saliva samples
  • Takes ~300 minutes for sample preparation and ~90 minutes for library preparation.
  • Requires no PCR
  • Is compatible with R10.4.1 flow cells

For Research Use Only

FOR RESEARCH USE ONLY

Descripción general

End-to-end method outlining sample extraction, library preparation, sequencing and data analysis. This protocol:

  • Uses genomic DNA extracted from human saliva samples
  • Takes ~300 minutes for sample preparation and ~90 minutes for library preparation.
  • Requires no PCR
  • Is compatible with R10.4.1 flow cells

For Research Use Only

Document version: HVSA_9216_v114_revA_22Jan2025

1. Aspectos generales (3)

Introducción al protocolo Ligation Sequencing Kit V14 (SQK-LSK114) (3)

Este protocolo describe cómo llevar a cabo la secuenciación de una muestra de ADN, utilizando el kit de secuenciación Ligation Sequencing Kit V14 (SQK-LSK114). Antes de empezar, se recomienda hacer un experimento de control con lambda para familiarizarse con la tecnología.

Para mayor información sobre la identificación de bases duplex, consulte el documento Kit 14 sequencing and duplex basecalling.

Proceso de secuenciación:

Preparación del experimento

Pasos:

  • Extraer el ADN y evaluar su longitud, cantidad y pureza. Los controles de calidad realizados durante el protocolo son fundamentales para garantizar el éxito del experimento.
  • Contar con el kit de secuenciación, el instrumental adecuado y los reactivos de otros fabricantes.
  • Descargar el programa MinKNOW para obtener y analizar los datos.
  • Comprobar que la celda de flujo tiene poros suficientes para realizar una buena secuenciación.

Preparación de la biblioteca

La tabla siguiente es un resumen de los pasos necesarios en la preparación de la biblioteca, incluidos la duración y las paradas opcionales.

Prep biblioteca Proceso Duración Parada opcional
Reparación del ADN y preparación de los extremos Reparar el ADN y preparar los extremos para ligar el adaptador 35 minutos Guardar a 4°C hasta el día siguiente
Ligación de los adaptadores y purificación Ligar los adaptadores suministrados en el kit a los extremos de ADN 20 minutos Guardar a 4°C durante un periodo breve o durante un uso repetido, como en la recarga de la celda de flujo
Guardar a -80°C si se va a usar una sola vez, tras un almacenamiento prolongado.
Se recomienda secuenciar la biblioteca tan pronto como se liguen los adaptadores.
Cebado y carga de la celda de flujo Cebar la celda de flujo y cargar la biblioteca de ADN en ella 5 minutos

Spanish LSK114 Workflow

Secuenciación y análisis

Pasos:

  • Empezar el proceso de secuenciación usando el programa MinKNOW, que obtendrá datos crudos del dispositivo y los convertirá en lecturas de bases identificadas.
  • Iniciar el programa EPI2ME y seleccionar un proceso de trabajo bioinformático para analizar los datos.
IMPORTANTE

Compatibility of this protocol

This protocol should only be used in combination with:

2. Equipment and consumables

Material
  • (FOR EXTRACTION) 2 ml of human saliva in a Genefix™ Saliva DNA/RNA Collection & Stabilisation tube (GFX-02) containing 2 ml of lysis buffer.
  • (FOR LIBRARY PREPARATION) 2 µg of SFE size selected and Megaruptor fragmented gDNA
  • Short Fragment Eliminator Expansion (EXP-SFE001)
  • Ligation Sequencing Kit V14 (SQK-LSK114) (kit de secuenciación por ligación V14)
  • Flow Cell Wash Kit (EXP-WSH004)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
Consumibles
  • Celda de flujo PromethION
  • Genefix™ Saliva DNA/RNA Collection & Stabilisation tube - 2ml (Isohelix, GFX-02)
  • Genefix™ Saliva-Prep 2 DNA Isolation Kit (Isohelix, GSPN)
  • Megaruptor 3 Shearing Kit (Diagenode, E07010003)
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • NEBNext® Companion Module v2 para Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7672S o E7672L) (1)
  • NEBNext FFPE Repair Mix (NEB M6630) (mezcla de reparación de ADN)
  • NEBNext FFPE DNA Repair v2 Module (NEB, E7360)
  • NEBNext Ultra II End Repair/dA-tailing Module (NEB E7546) (Módulo de reparación de extremos/Adición de dA)
  • Salt-T4® DNA Ligase (NEB, M0467)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Etanol al 80 %, recién preparado con agua sin nucleasas
  • Isopropanol, 100% (Fisher, 10723124)
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851)
  • Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
  • Tubos de ensayo Qubit™ (Invitrogen Q32856)
  • 15 ml Falcon tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Tubos de PCR de pared fina (0,2 ml)
Instrumental
  • PromethION device
  • PromethION Flow Cell Light Shield
  • Mezclador Hula (mezclador giratorio suave)
  • Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
  • Heating block
  • Incubator or water bath
  • Microcentrífuga
  • Mezclador vórtex
  • Termociclador
  • Centrifuge and rotor suitable for 15 ml Falcon tubes
  • Megaruptor 3 (Diagenode, B06010003)
  • 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
  • Fluorímetro Qubit (o equivalente para el control de calidad)
  • Agilent Femto Pulse System (or equivalent for read length QC)
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:

  • 2 ml of human saliva in a Genefix™ Saliva DNA/RNA Collection & Stabilisation tube (GFX-02) containing 2 ml of lysis buffer.

Input requirements per sample for the library preparation:

  • 2 µg of SFE size selected and Megaruptor 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.

CONSEJO

Eppendorf tube orientation in centrifuge

For all centrifugation steps, ensure that tubes are loaded into the centrifuge with the hinge side of the tube facing outwards. This will assist in visual identification of the pellet.

Ensure gentle handling when removing the tubes from the centrifuge to avoid dislodging the pellet.

Eppendorf tube hinge into centrifuge image

CONSEJO

Recomendamos utilizar el módulo de acompañamiento Companion Module v2 para Oxford Nanopore Technologies® Ligation Sequencing (ref. E7672S or E7672L) de NEBNext®, que contiene los reactivos necesarios para utilizar junto al Ligation Sequencing Kit.

La versión anterior, NEBNext® Companion Module para Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S o E7180L) también es compatible, pero el modelo recomendado v2, ofrece una ligadura y adición de dA más eficaces.

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

El adaptador incluido en este kit, Ligation Adapter (LA), no es intercambiable con otros adaptadores de secuenciación.

Contenido del kit Ligation Sequencing Kit V14 (SQK-LSK114)

Nota: Hemos cambiando el formato de nuestros kits; hemos sustituido algunos de los viales de un solo uso por botellas de mayor contenido.

Formato de tubos monouso SQK-LSK114 v2 ES

Formato en botella SQK-LSK114 v3 ES

Nota: este producto contiene un reactivo, AMPure XP, fabricado por Beckman Coulter Inc., que puede conservarse con el kit a -20 °C sin perjudicar su estabilidad.

Nota: la muestra de control de ADN (DCS) es un amplicón estándar de 3,6 kb, que mapea el extremo 3' del genoma Lambda.

3. Purification of gDNA from 2 ml of human saliva

Material
  • 4 ml saliva sample in Genefix Saliva DNA/RNA Collection & Stabilisation tube (GFX-02) (2 ml of human saliva and 2 ml of lysis buffer)
Consumibles
  • Genefix™ Saliva-Prep 2 DNA Isolation Kit (Isohelix, GSPN)
  • Genefix™ Saliva DNA/RNA Collection & Stabilisation tube - 2ml (Isohelix, GFX-02)
  • Absorbent material e.g. paper towel or tissues
  • 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)
  • 15 ml Falcon tubes
  • 2 ml Eppendorf DNA LoBind tubes
Instrumental
  • Centrifuge and rotor suitable for 15 ml Falcon tubes
  • Incubator or water bath
  • Mezclador vórtex
  • Microfuge
  • Fluorímetro Qubit (o equivalente para el control de calidad)
  • Ice bucket with ice
  • Timer
  • Wide-bore pipette tips
  • P1000 pipette and tips
  • Pipeta y puntas P200
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • P2 pipette and tips
Equipo opcional
  • Agilent Femto Pulse System (or equivalent for read length QC)

Vortex the GeneFix™ collection tube containing your saliva sample for ten seconds to mix well.

Add 40 µl Proteinase K solution to the tube containing your saliva sample and vortex to mix thoroughly.

Incubate the tube at 60°C for 1 hour.

Transfer the sample solution into a fresh 15 ml conical centrifuge tube (falcon tube).

Add 4 ml of SPN buffer to the 15 ml falcon tube containing the sample, and vortex well to mix thoroughly.

Centrifuge the tube at 3,000 x g for 30 minutes.

Carefully pour off the supernatant, taking care not to disturb the pellet.

Centrifuge the tube again at 3,000 x g for 1 minute.

Using a pipette, carefully remove all remaining supernatant liquid, taking care not to disturb the DNA pellet.

Note: It is important to remove all of the supernatant liquid.

Add 400 µl TE buffer to the sample tube, and vortex to fully resuspend the sample and mix thoroughly.

Incubate the resuspended sample at room temperature for at least 5 minutes, allowing the DNA to re-hydrate.

Transfer the full volume of resuspended sample to a clean 2 ml Eppendorf tube.

Centrifuge the sample tube at 12,000 x g for 15 minutes to remove any undissolved particulates.

Remove the supernatant and transfer it to a clean 2 ml Eppendorf tube, being careful not to disturb the pellet.

Note: The pellet is discarded.

Add 400 µl of SLS buffer to the tube containing the sample supertant. Vortex to mix thoroughly.

Add 800 µl SPN buffer to the sample supertant. Vortex to mix thoroughly.

Centrifuge the tube at 12,000 x g for 10 minutes.

Carefully pour off the supernatant, taking care not to disturb the pellet.

Centrifuge the tube again at 12,000 x g for 1 minute.

Using a pipette, carefully remove all remaining supernatant liquid, taking care not to disturb the DNA pellet.

Note: The DNA pellet may be difficult to see at this point. Please take care when removing the supernatant.

It is important to remove all of the supernatant liquid.

Add 103 µl of TE buffer to the tube containing your pelleted sample, and mix by gently flicking the tube.

Incubate the tube in a heat block at 37°C for 30 minutes. Gently agitate the solution by flicking every 5 minutes to aid with resuspension.

Gently mix the tube contents by pipetting up and down using a wide-bore tip.

IMPORTANTE

If your Qubit measurements are not consistent, this could indicate that the DNA has not been homogeneously resuspended.

If this occurs, we recommend performing additional mixing/agitation steps and increasing incubation and elution time:

  • Mix your sample again with a wide-bore tip
  • Elute your sample at 50°C for 1 hour in a thermomixer set to 300 rpm.

Quantify your sample three times using the Qubit dsDNA BR Assay Kit. Ensure the replicate Qubit measurements are consistent before continuing to the next step.

We suggest taking a reading from the top, middle and bottom of the sample tube to test for homogeneity.

Note: Approximately 3–20 µg of gDNA is expected following sample extraction.
Expected Qubit measurements of 30–200 ng/μl.

MEDIDA OPCIONAL

Your extracted gDNA can also be analysed using Femto Pulse (Agilent) to check the size and quality.

Frag length profile saliva extract hum var oct2024

Example fragment length profile of gDNA extracted from salvia collected in a Genefix™ Saliva DNA/RNA Collection & Stabilisation tube - 2ml and extracted using the Genefix™ Saliva-Prep 2 DNA Isolation Kit.

FIN DEL PROCESO

Take your extracted gDNA forward into the size selection of gDNA step of this protocol. Alternatively, your sample can be stored at 4°C overnight.

4. Size selection of gDNA using SFE Buffer

Material
  • 10 µg extracted human gDNA
  • Short Fragment Eliminator Expansion (EXP-SFE001)
Consumibles
  • TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Etanol al 80 %, recién preparado con agua sin nucleasas
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Tubos de ensayo Qubit™ (Invitrogen Q32856)
  • 1.5 ml Eppendorf DNA LoBind tubes
Instrumental
  • Centrifuge
  • Heating block
  • Mezclador vórtex
  • Microfuge
  • Fluorímetro Qubit (o equivalente para el control de calidad)
  • Ice bucket with ice
  • Timer
  • Wide-bore pipette tips
  • P1000 pipette and tips
  • Pipeta y puntas P200
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • P2 pipette and tips
Equipo opcional
  • Agilent Femto Pulse System (or equivalent for read length QC)
CONSEJO

Though we recommend starting with 10 µg, we have found that a minimum of 3 µg input DNA is still sufficient to proceed with size selection using SFE buffer.

Size selection is not recommended if gDNA recovery is below 3 µg, as recovery will be insufficient for sequencing.

If less than 3 µg of gDNA is recovered, size selection can be omitted; however, this will typically result in read length N50s between 6-13 kb, negatively impacting the accuracy and reliability of variant identification.


Read length profile saliva 9kb

Read length profile for a 9 kb N50 library that was prepared without the use of SFE size selection due to the sample having a low extraction yield (considered here as 1 – 3 ug). These samples can still be sequenced, but will contain more short reads relative to a sample that used SFE size selection. This will negatively impact the accuracy and reliability of variant identification.

In a 1.5 ml Eppendorf DNA LoBind tube, prepare 10 µg DNA in 100 µl of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) to a final concentration of ~100 ng/μl.

Label your sample tube as "tube 1" for tracking.

Ensure the Short Fragment Eliminator (SFE) buffer is at room temperature. Then pipette mix the buffer thoroughly, at least 10-15 times, using a wide bore pipette tip.

Add twice the volume (200 µl) of Short Fragment Eliminator (SFE) buffer to the DNA sample.

Note: The increased volume of SFE used in this method (compared to the 100 µl described in our other methods using SFE) has been shown to provide optimal recovery for saliva samples. More information on this can be found in our Short Fragment Eliminator Kit (EXP-SFE-001) know-how document.

Using a wide-bore tip set to 200 μl, slowly and gently mix 10-15 times to thoroughly mix the SFE buffer with the sample.

Ensure that the solution is homogenous before proceeding to the next step.

IMPORTANTE

The pellet might not be visible after centrifugation, so it's essential to maintain the correct orientation of the tube to avoid accidentally disturbing the pellet in the following steps.

Place the tube in the centrifuge with the tube hinge facing outward, and add the appropriate tubes to balance the centrifuge.

Ensure proper tube orientation before starting and that the centrifuge is at the correct temperature prior use.

Centrifuge the sample at 10,000 x g at 22°C for 30 minutes.

Note: Ensure that the centrifuge is at the correct temperature prior use.

CONSEJO

The pellet may not be visible but it will be located on the side of the tube that was facing outwards during centrifugation. We recommend not removing the full volume of the supernatant and leaving ~15–20 µl as this will be washed out in subsequent steps.

Using a narrow bore pipette tip, slowly and carefully remove most of the supernatant (leaving ~ 15-20 μl behind) by aspirating from the opposite side of the tube from the location of the pellet, and store the removed supernatant in a clean 1.5 ml Eppendorf tube. Take care not to disturb the DNA pellet.

Note: Ensure you keep the volume of the aspirated supernatant and store it in a separate 1.5 ml Eppendorf tube. This is used in subsequent steps for re-centrifugation to maximise yield.

Label your tube with the saved supernatent as "tube 2" for tracking.

Repeat the centrifugation and DNA pelleting steps for the retained supernatant in "tube 2" to maximise DNA yield:

1. Place "tube 2" in the centrifuge with the tube hinge facing outward, and add the appropriate tubes to balance the centrifuge.

2. Centrifuge "tube 2" at 10,000 x g at 22°C for 30 minutes.
Note: Ensure that the centrifuge is at the correct temperature prior use.

3. Using a narrow bore pipette tip, slowly and carefully remove most of the supernatant (leaving ~ 15-20 μl behind) by aspirating from the opposite side of the tube from the location of the pellet.
Note: The supernatant from the secondary centrifugation step will contain only short fragments of gDNA and can be discarded.

Perform an ethanol wash to your sample tubes containing your DNA pellets in ~15–20 μl ("tube 1" and "tube 2").

To each tube:

  • slowly add 200 μl of freshly prepared 80% ethanol to the tube, without disturbing the pellet (we recommend dispensing on the opposite side of the tube as your pellet).
  • centrifuge the tubes at 10,000 x g for 3 minutes ensuring the same tube orientation used in for the previous centrifuge step (hinge facing outwards).
  • Using a narrow bore tip, slowly and gently aspirate the supernatant from the opposite side of the tubes, taking care not to disturb the pellet.

Note: After the first ethanol wash, the pellet may become more visible (as a white pellet at the bottom of the hinge-side of the tube):

  • If the pellet is visible, then carefully discard as much of the ethanol as you can.
  • If the pellet is not visible, leave behind ~20 μl volume of supernatant to avoid inadvertently aspirating or disturbing the pellet.

Note: To protect against loss of DNA by inadvertent aspiration of pelleted DNA, the aspirated supernatant should be stored in a separate 1.5 ml Eppendorf tube, labelled accordingly for tracking and stored in the fridge.

Repetir el paso anterior. (2)

Note: After the second ethanol wash, the pellet in the tube should be clearly visible in most cases and the residual ethanol can be carefully discarded using a 10 μl pipette, taking care not to disturb the pellet.

Note: To protect against loss of DNA by inadvertent aspiration of pellet DNA, the aspirated supernatant should be stored in a separate 1.5 ml Eppendorf tube, labelled accordingly for tracking and stored in the fridge.

Elute the gDNA from each sample tube ("tube 1" and "tube 2").

  • Add 50 μl of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) to each tube.
  • Pipette mix using a wide-bore tip.

Incubate the tubes in a in a thermomixer set to 300 rpm at 50°C for 30 minutes. Gently agitate the solutions by flicking every 5 minutes to aid with resuspension.

Optional: If necessary, the pellet can be left overnight to elute at room temperature.

Gently mix the tubes contents by pipetting up and down using a wide-bore tip, and combine the contents of "tube 1" and "tube 2" prior to downstream quantification and processing.

Quantify your sample three times using the Qubit dsDNA BR Assay Kit. Ensure the replicate Qubit measurements are consistent before continuing to the next step.

We suggest taking a reading from the top, middle and bottom of the sample tube to test for homogeneity.

Note: The readings should be consistent. If they are not, consider:

  • additional mixing steps/agitation of the sample with a wide-bore tip.
  • additional mixing and elution steps in a thermomixer set to 300 rpm at 50°C for 2 hours.

Note: You should expect to recover >2000 ng of gDNA following size selection.

FIN DEL PROCESO

Take your SFE size selected gDNA forward into the fragmentation step of this protocol. Alternatively, your sample can be stored at 4°C overnight.

5. gDNA fragmentation using the Megaruptor

Material
  • 2 µg of SFE size selected gDNA
Consumibles
  • Megaruptor 3 Shearing Kit (Diagenode, E07010003)
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
  • Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
  • 1.5 ml Eppendorf DNA LoBind tubes
Instrumental
  • Megaruptor 3 (Diagenode, B06010003)
  • Microcentrífuga
  • Agilent Femto Pulse System (or equivalent for read length QC)
  • Ice bucket with ice
  • Timer
  • Wide-bore pipette tips
  • P1000 pipette and tips
  • Pipeta y puntas P200
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • P2 pipette and tips

Preparación del ADN en agua sin nucleasas: (4)

  1. Transferir 1 μg (o 100-200 fmol) de muestra de ADN a un tubo de 1,5 ml Eppendorf DNA LoBind.

  2. Ajustar el volumen a un total de 47 μl con agua sin nucleasas.

  3. Mezclar minuciosamente con la pipeta o golpear el tubo suavemente con el índice.

  4. Centrifugar brevemente

Transfer the sample tube to the Megaruptor 3, ensuring the instrument is appropriately balanced according to the manufacturers instructions.

Note: Ensure the Megaruptor 3 Hydropore-Syringes are screwed tight before inserting into the Megaruptor.

Ensure no bubbles are present in the sample, and visually confirm that the syringe is immersed 2/3rd of the way into the sample volume.

Setup the shearing parameters on the Megaruptor 3 device as follows:

Megaruptor 3 setting
Shearing speed 27
Volume 90 μl
Concentration 20 ng/ul

Begin the shearing of DNA using the Megaruptor 3.

Quantify your sample using the Qubit dsDNA BR Assay Kit.

Note: Approximately 2 µg of gDNA is expected following shearing.
Expected Qubit measurements of ~22.22 ng/μl.

Assess the fragmented gDNA for fragment size using Femto Pulse (Agilent).

Frag length profile saliva extract Megarruptor1 hum var oct2024

Example fragment length profile of gDNA extracted, size selected and fragmented using Megaruptor 3.

The SFE size selection removes short DNA fragments from the sample, whilst Megaruptor fragmentation reduces the fragment length profile to a size range between 10 kb and 80 kb, centred around approximately 20 kb.

Frag length profile saliva extract Megarruptor2 hum var oct2024

Overlay of fragment length profiles of a) gDNA extracted using the Genefix™ Saliva-Prep 2 DNA Isolation Kit. b) gDNA extracted and size selected using SFE buffer. c) gDNA extracted, size selected and fragmented using Megaruptor 3 (MR).

FIN DEL PROCESO

Take your SFE size selected and Megaruptor fragmented gDNA forward into the library preparation section of this protocol. Alternatively, your sample can be stored at 4°C overnight.

6. Reparación del ADN y preparación de los extremos (3)

Material
  • 2 µg of SFE size selected and Megaruptor fragmented gDNA
  • AMPure XP Beads (AXP)
Consumibles
  • NEBNext® FFPE DNA Repair Mix (M6630), del Companion Module v2 (NEB, E7672S o E7672L) de NEBNext®
  • NEBNext® FFPE DNA Repair Buffer v2 (E7363), del Companion Module v2 (NEB, E7672S o E7672L) de NEBNext®
  • NEBNext® Ultra II End Prep Enzyme Mix (E7646), del Companion Module v2 (NEB, E7672S o E7672L) de NEBNext®
  • Kit de ensayo Qubit dsDNA HS (Invitrogen Q32851)
  • 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)
CHECKPOINT

Verificar la celda de flujo

Antes de empezar a preparar la biblioteca, recomendamos verificar la celda de flujo para comprobar que tiene poros suficientes para realizar un buen experimento.

Las instrucciones de comprobación de la celda de flujo están disponibles en el protocolo de MinKNOW.

Preparar los reactivos NEB siguiendo las instrucciones del fabricante y poner en hielo.

Para obtener un rendimiento óptimo, NEB recomienda lo siguiente:

  1. Descongelar todos los reactivos en hielo.

  2. Golpear suavemente los tubos de los reactivos con el índice o invertirlos, a fin de mezclarlos bien.
    Nota: No mezclar en vórtex las mezclas FFPE DNA Repair Mix, ni Ultra II End Prep Enzyme Mix.

  3. Centrifugar los tubos antes de abrirlos.

  4. Mezclar en vórtex los tampones FFPE DNA Repair Buffer v2 o FFPE DNA Repair Buffer y Ultra II End Prep Reaction Buffer, a fin de mezclarlos bien.

    Nota: Es posible que los tampones tengan un precipitado blanco. Si ello ocurre, dejar que la mezcla se ponga a temperatura ambiente y mezclar el tampón con la pipeta varias veces para romper el precipitado; a continuación, mezclar rápido en vórtex.

  5. 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)

  1. Transferir 1 μg (o 100-200 fmol) de muestra de ADN a un tubo de 1,5 ml Eppendorf DNA LoBind.

  2. Ajustar el volumen a un total de 47 μl con agua sin nucleasas.

  3. Mezclar minuciosamente con la pipeta o golpear el tubo suavemente con el índice.

  4. Centrifugar brevemente

En un tubo de PCR de pared fina (0,2 ml), mezclar lo siguiente: (2)

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, incubate the reaction at 20°C for 5 minutes, then 65°C for 5 minutes and hold at 4°C.

Resuspender las microesferas magnéticas AMPure XP Beads (AXP) agitándolas en vórtex.

Transferir la muestra de ADN a un tubo Eppendorf DNA Lobind de 1,5 ml.

Add 100 µl of resuspended the AMPure XP Beads (AXP) to the 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 600 µl of fresh 80% ethanol in nuclease-free water.

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. (1)

Keep the tube on the magnet and wash the beads with 250 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

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 precipitado 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.

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.

CHECKPOINT

Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit. (3)

Note: You should expect to recover between 1300–1600 ng.

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. (2)

7. Adapter ligation and clean-up

Material
  • 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)
  • AMPure XP Beads (AXP)
  • Elution Buffer (EB) (tampón de elución) del kit de Oxford Nanopore
Consumibles
  • Salt-T4® DNA Ligase (NEB, M0467)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Kit de ensayo Qubit dsDNA HS (Invitrogen Q32851)
  • Tubos de ensayo Qubit™ (Invitrogen Q32856)
Instrumental
  • Gradilla magnética
  • Microcentrífuga
  • Mezclador vórtex
  • Pipeta y puntas P1000
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • Fluorímetro Qubit (o equivalente para el control de calidad)
IMPORTANTE

Aunque la ligasa recomendada de otros fabricantes se suministra con su propio tampón, la eficiencia del adaptador, Ligation Adapter (LA), es mayor cuando se usa el tampón Ligation Buffer (LNB) suministrado en el kit Ligation Sequencing Kit.

Centrifugar los viales Ligation Adapter (LA) y Quick T4 Ligase y poner en hielo. (1)

Descongelar el vial Ligation Buffer (LNB) a temperatura ambiente, centrifugar y mezclar con la pipeta. Debido a su viscosidad, la agitación en vórtex de este tampón es ineficaz. Tras descongelar y mezclar, colocar en hielo inmediatamente.

Descongelar el vial Elution Buffer (EB) a temperatura ambiente, agitar en vórtex, centrifugar y colocar en hielo.

Thaw the Long Fragment Buffer (LFB) at room temperature and mix by vortexing. Then spin down and place on ice.

En un tubo Eppendorf DNA LoBind de 1,5 ml mezclar en el siguiente orden: (1)

Entre cada adición, mezclar con la pipeta de 10 a 20 veces.

Reactivo Volumen
Muestra de ADN del paso anterior 60 µl
Ligation Buffer (LNB) 25 µl
NEBNext Quick T4 DNA Ligase 10 µl
Ligation Adapter (LA) 5 µl
Total 100 µl

Mezclar pipeteando con suavidad y centrifugar brevemente la reacción para asegurarse de que se mezcla completamente.

Incubar la reacción durante 10 minutos a temperatura ambiente.

Resuspender las microesferas magnéticas AMPure XP Beads (AXP) agitándolas en vórtex.

Añadir 40 μl de microesferas magnéticas resuspendidas AMPure XP Beads (AXP) a la reacción y mezclar dando suaves golpes al tubo con el dedo.

Incubar en el mezclador Hula (o mezclador giratorio suave) durante 5 minutos a temperatura ambiente.

Centrifugar la muestra y precipitar en un imán. Dejar el tubo en el imán y retirar el sobrenadante con una pipeta.

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 65 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at 37°C.

Precipitar las microesferas en un imán, durante al menos 1 minuto, hasta que el eluido se vuelva claro e incoloro. (1)

Remove and retain 65 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. (3)

Note: You should expect to recover 400–800 ng of adapter ligated library in a volume of 64 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.

8. Priming and loading the PromethION Flow Cell

Material
  • Sequencing Buffer (SB)
  • Library Beads (LIB)
  • Flow Cell Tether (FCT)
  • Flow Cell Flush (FCF)
Consumibles
  • Celda de flujo PromethION
  • Tubos de 1,5 ml Eppendorf DNA LoBind
Instrumental
  • Dispositivo PromethION 24/48
  • PromethION Flow Cell Light Shield
  • P1000 pipette and tips
  • Pipeta y puntas P200
  • 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:

  1. Place the flow cell flat on the metal plate.

  2. Slide the flow cell into the docking port until the gold pins or green board cannot be seen.

J2068 FC-into-P2-animation V5

For the PromethION 24/48, load the flow cell(s) into the docking ports:

  1. Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
  2. Press down firmly onto the flow cell and ensure the latch engages and clicks into place.

Step 1a V3

Step 1B

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.

Screenshot 2021-04-08 at 12.08.37

Slide the inlet port cover clockwise to open.

Prom loading 2

IMPORTANTE

Tenga cuidado a la hora de extraer solución amortiguadora de la celda de flujo. No retire más de 20-30 μl y asegúrese de que la solución 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:

  1. Set a P1000 pipette tip to 200 µl.
  2. Insert the tip into the inlet port.
  3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Step 3 v1

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.

Step 4 v1

Mezclar minuciosamente con la pipeta el contenido del vial Library Beads (LIB).

IMPORTANTE

El vial Library Beads (LIB) contiene microesferas en suspensión. Las microesferas sedimentan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación recomendamos utilizar Library Beads (LIB). El reactivo Library Solution (LIS) está disponible en caso de utilizar bibliotecas 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.

Step 5 v1

Mezclar la biblioteca suavemente con la pipeta, justo antes de cargar.

Load 200 µl of library into the inlet port using a P1000 pipette.

Step 6 v1

Close the valve to seal the inlet port.

Step 7 V2

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:

  1. 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.
  2. Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

J2264 - Light shield animation PromethION Flow Cell 8a FAW

J2264 - Light shield animation PromethION Flow Cell 8b FAW

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 ~36 hours of sequencing.

Follow the instructions in the washing and reloading a PromethION Flow Cell section of this protocol.

9. Washing and reloading a PromethION Flow Cell

Material
  • Adapter ligated DNA library (from previous step)
  • Flow Cell Wash Kit (EXP-WSH004)
  • 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 ~36 hours of sequencing.

For this method, the flow cell is washed after ~36 hours of sequencing to restore pores to ensure efficient data acquisition. For this reason, enough library was generated for 2 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 asterisk indicates the flow cell wash and reloads.

Channel state saliva extract hum var oct2024

Figure 1. Channel state over a 72 hour run. The flow cell wash is incorporated into the method to restore blocked pores, to allow continuous data acquisition. Red asterisk denotes when a flush was performed.


cummulative data output saliva extract hum var oct2024

Figure 2. Cumulative sequencing data output, over a 72 hour run. Red asterisk denotes when a flush was performed.


Sequencing read length profile saliva extract hum var oct2024

Figure 3. Read length profile for a 20 kb N50 library. The approximately gaussian shape is characteristic of gDNA that has undergone SFE size selection and Megaruptor shearing. The short distribution of reads at ~5kb is due to premature termination of reads at the mux scan.

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:

  1. Close the inlet port.
  2. 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.

Step 2 V2

IMPORTANTE

Tenga cuidado a la hora de extraer solución amortiguadora de la celda de flujo. No retire más de 20-30 μl y asegúrese de que la solución 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:

  1. Set a P1000 pipette to 200 µl
  2. Insert the tip into the inlet port
  3. Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Step 3 v1

Slowly load 200 µl of the prepared flow cell wash mix into the inlet port, as follows:

  1. Using a P1000 pipette, take 200 µl of the flow cell wash mix
  2. Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
  3. 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.
  4. 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:

  1. Using a P1000 pipette, take 200 µl of the flow cell wash mix
  2. Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
  3. 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.

Step 7 V2 edited to step 5

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:

  1. Ensure the inlet port is closed.
  2. 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.

Prom loading 2

IMPORTANTE

Tenga cuidado a la hora de extraer solución amortiguadora de la celda de flujo. No retire más de 20-30 μl y asegúrese de que la solución 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:

  1. Set a P1000 pipette tip to 200 µl.
  2. Insert the tip into the inlet port.
  3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Step 3 v1

Slowly load 500 µl of the priming mix into the inlet port, as follows:

  1. Using a P1000 pipette, take 500 µl of the priming mix
  2. Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
  3. 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.

Step 5 v1

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 minuciosamente con la pipeta el contenido del vial Library Beads (LIB).

IMPORTANTE

El vial Library Beads (LIB) contiene microesferas en suspensión. Las microesferas sedimentan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación recomendamos utilizar Library Beads (LIB). El reactivo Library Solution (LIS) está disponible en caso de utilizar bibliotecas 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:

  1. Ensure the inlet port is closed.
  2. 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.

Prom loading 2

IMPORTANTE

Tenga cuidado a la hora de extraer solución amortiguadora de la celda de flujo. No retire más de 20-30 μl y asegúrese de que la solución 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:

  1. Set a P1000 pipette tip to 200 µl.
  2. Insert the tip into the inlet port.
  3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Step 3 v1

Slowly load 500 µl of the priming mix into the inlet port, as follows:

  1. Using a P1000 pipette, take 500 µl of the priming mix
  2. Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
  3. 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.

Step 5 v1

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:

  1. Close the inlet port.
  2. 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.

Prom loading 2

IMPORTANTE

Tenga cuidado a la hora de extraer solución amortiguadora de la celda de flujo. No retire más de 20-30 μl y asegúrese de que la solución 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:

  1. Set a P1000 pipette tip to 200 µl.
  2. Insert the tip into the inlet port.
  3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Step 3 v1

Mezclar la biblioteca suavemente con la pipeta, justo antes de cargar.

Load 200 µl of library into the inlet port using a P1000 pipette.

Step 6 v1

Close the valve to seal the inlet port.

Step 7 V2

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:

  1. 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.
  2. Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

J2264 - Light shield animation PromethION Flow Cell 8a FAW

J2264 - Light shield animation PromethION Flow Cell 8b FAW

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.

10. Data acquisition and basecalling

How to start sequencing

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software.

We recommend basecalling with the high accuracy (HAC) basecaller in real-time with BAM selected as output type using the P2 Solo or P24/P48 device.

You must generate a BAM file from your sequening run, as this is required for input into the wf-human-variation workflow.

Refer to the links below containing the detailed instructions for setting up the device and sequencing run:

Below are the recommended sequencing parameters for MinKNOW.

MinKNOW settings for human saliva sample variant workflow on PromethION

We recommend setting the run time to 100 hours to accommodate for the flow cell wash, using the modified bases option for basecalling and ensuring a BAM output is selected. All other sequencing parameters can be kept to their default settings. Below are our current recommendations:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-PRO114M

Sample ID: [user defined]

Kit

Kit selection: Ligation Sequencing Kit (SQK-LSK114)

Run configuration

Sequencing and analysis

Basecalling: On [default] Modified bases: On with '5mC & 5hmC CG contexts' selected Model: High-accuracy basecalling (HAC) [default]

Barcoding: Disabled [default]

Alignment: Off [default]
We do not currently recommend live alignment during sequencing, as it can slow down system processing.

Adaptive sampling: Off [default]

Advanced options Active channel selection: On [default] Time between pore scans: 1.5 [default] Reserve pores: On [default]

Data targets

Run limit: 100 hours [default]

Output

Output format .POD5: On [default] .FASTQ: On [default] .BAM: On

Filtering: On [default] Qscore: 9 [default] Minimum read length: 200 bp [default]

IMPORTANTE

We do not recommend live alignment during sequencing, as it can slow down system processing.

You can align your BAM file post-sequencing by following one of the methods below:

Aligning the BAM file in MinKNOW Aligning the BAM file during the wf-human-variation workflow
Align the BAM output after live basecalling in MinKNOW. This will prevent slowing down your sytems processing.

The aligned BAM file can be used as your file input in the wf-human-variation workflow.

Using mapped BAM as input, the workflow will take 1-2 hours.		 You can provide a reference genome along with the unaligned BAM file during the wf-human-variation workflow set-up.

Using an unmapped BAM is used as input, the workflow will take approximately 5-8 hours.

Further information is available in the Downstream analysis section of this protocol.

11. Downstream analysis

Analysis of human saliva DNA sequence data

For the analysis of human saliva DNA sequence data, we recommend the wf-human-variation workflow. This end-to-end software pipeline is implemented using the Nextflow workflow language and implements methods for the calling of single nucleotide polymorphisms (SNPs), structural variants (SVs), and for reporting DNA methylation information.

The wf-human-variation workflow is best run from the BAM file produced by MinKNOW when the modified base model for basecalling is selected. If sequence read mapping to the reference genome is not performed by MinKNOW, we recommend to perform the basecalling using the wf-basecalling workflow. Ensure you save the outputs in BAM format by providing the --output_bam option.

The tools below are used in the analysis workflow and can be run in isolation or together:

  1. Sniffles2 calls SVs and file output include an HTML report of QC metrics and VCF format list of variants and their quality scores.

  2. Clair3 calls SNPs and file output includes an HTML report of QC metrics and VCF format list of variants and their quality scores.

  3. modkit extracts methylation information from the provided BAM file which is summarised in a BEDmethyl format file.

The wf-human-variation workflow is preconfigured using appropriate parameters and requires tuning only for the choice of reference genome and Clair3 model. Please see the project’s documentation for further details.

The results from the wf-human-variation workflow can be further explored by viewing in a track-based genome browser such as IGV can be assessed for known pathogenicity through tertiary analysis software.

EPI2ME analysis workflow

The wf-human-variation workflow is intended to be run using the Nextflow software.

We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a Graphical User Interfaces (GUI). EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis. The collection of all our available EPI2ME workflows can be found here.

For new users, the quick start guide can be found here outlining how to use this interface.

IMPORTANTE

Compute requirements for the wf-human-variation workflow on EPI2ME

Recommended requirements Minimum requirements
CPUs = 32 CPUs = 16
Memory = 128GB Memory = 32GB

Approximate run time: Variable depending on whether it is targeted sequencing or whole genome sequencing, as well as coverage and the individual analyses requested. For instance, a 90X human sample run (options: --snp --sv --mod --str --cnv --phased --sex male) takes less than 8h with recommended resources.

ARM processor support: False

MEDIDA OPCIONAL

The wf-human-variation workflow can also be run using the command line interface (CLI)

Please see the Github page for further details.

Note: We only recommend the command line interface (CLI) for experienced users.

Open the EPI2ME app using the desktop shortcut.

On the landing page, open the workflow tab on the left-hand sidebar.

1 landing page EPI2ME

Navigate to the Available workflows tab and click on wf-human-variation option.

2 wf human var

Click install.

3 install human var

Navigate to the Installed tab and click on the installed wf-human-variation workflow.

4 wf human var select

MEDIDA OPCIONAL

If the workflow was already installed, check for updates by clicking 'Update workflow'.

We recommend running the latest version of our workflows for the best results.

5 wf human var update

Click on Run this workflow to open the launch wizard.

6 human var run

Select the environment you are running the workflow from:

7 human var run II

Click on the sub-workflow(s) you want to run in the wf-human-variation analysis.

8 wf human var subworkflows

You must have at least one of the sub-workflows selected to proceed with analysis.

9 wf human var subworkflows selected

Note: For more information on the sub-workflows click on the "Expand" option in the platform, or visit our online EPI2ME documentation.

Navigate to the 'Main options' tab to assign a 'Sample name' as an identifier in workflow outputs.

10 wf human var sample name

IMPORTANTE

The wf-human-variation workflow uses sequencing data in the form of a single BAM file or a folder of BAM files.

The BAM files used as an input can be aligned or unaligned:

Aligning the BAM file in MinKNOW (prior to the wf-human-variation workflow) Aligning the BAM file during the wf-human-variation workflow (during the wf-human-variation workflow)
Align the BAM output after live basecalling in MinKNOW. This will prevent slowing down your sytems processing.

The aligned BAM file can be used as your file input in the wf-human-variation workflow.

For more information on post-run alignment in MinKNOW please visit our MinKNOW protocol.

Using mapped BAM as input, the workflow will take 1-2 hours.		 You can provide a reference genome along with the unaligned BAM file during the wf-human-variation workflow set-up.

Using an unmapped BAM is used as input, the workflow will take approximately 5-8 hours.

In the 'Main options' upload your sequencing data in the form of a single BAM file or a folder of BAM files.

11 wf human var BAM input

MEDIDA OPCIONAL

If you have an unaligned BAM file as input, in the 'Main options' upload your reference genome in FASTA format.

12 wf human var reference FASTA

Click Launch workflow.

Ensure all parameter options have green ticks.

13 wf human var launch

Once the wf-human-variation workflow finishes, a report will be produced alongside output files.

14 wf human var outputs

wf-human-variation workflow outputs

The primary workflow outputs include:

  • gzipped VCF file containing the SNPs in the dataset from --snp
  • gzipped VCF file containing the SVs in the dataset from --sv
  • gzipped bedMethyl file aggregating modified base counts from --mod
  • HTML report detailing the primary findings of the workflow for QC metrics, and SNP and SV calling
  • If an unaligned BAM file was provided, the workflow will ouput a CRAM file containing the alignments used to make the downstream variant calls.

The secondary workflow outputs:

  • mosdepth ouputs include:
    • {sample_name}.mosdepth.global.dist.txt: a cumulative distribution indicating the proportion of total bases for each and all reference sequences
    • {sample_name}.regions.bed.gz: the mean coverage for each region in the provided BED file
    • {sample_name}.thresholds.bed.gz: the number of bases in each region that are covered at or above each threshold value (1, 10, 20, 30X)

  • bamstats ouputs include:
    • {sample_name}.readstats.tsv.gz: a gzipped TSV summarising per-alignment statistics produced by bamstats
    • {sample_name}.ftagstat.tsv: a text file with summary alignment statistics for each reference sequence

wf-human-variation workflow tips

It is possible to phase SNPs, SVs and modified bases by providing the --phased option.

To improve the accuracy of SV calling, specify a suitable tandem repeat BED for your reference with --tr_bed.

Aggregation of methylation calls with --mod requires data to be basecalled with a model that includes base modifications, providing the MM and ML BAM tags. To do so on MinKNOW, ensure 'Modified bases' option is selected during basecalling set up, with the '5mC' model selected.

Ensure to retain the input reference when basecalling or alignment is performed as CRAM files cannot be read without the corresponding input reference.

For a full list of available basecalling models, refer to the Dorado documentation.

12. 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.

13. Issues during DNA extraction and library preparation

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 chat Live Support de la comunidad Nanopore.

Low sample quality

Observation Possible cause Comments and actions
Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2) The DNA extraction method does not provide the required purity The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

Consider performing an additional AMPure bead clean-up step.

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. SPRI cleanup
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.

14. 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 chat Live Support de la comunidad Nanopore.

Menos poros al inicio de la secuenciación que tras verificar la celda de flujo

Observaciones Posibles causas 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, y antes de acondicionarla, es imprescindible quitar las burbujas que haya cerca del puerto de purgado. 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 podría haber dañado o bloqueado los poros El número de poros resultante tras la evaluación 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 podría 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

Observaciones Posibles causas 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 cargada a 4 °C y contacte con el servicio de asistencia técnica para recibir instrucciones 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 For the human genome sequencing protocols, 200-300 ng of good quality library should be loaded on to an R10.4.1 flow cell to keep pore occupancy high.
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 SQK-LSK114 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 (FCT tube). Make sure FCT was added to FCF before priming.

Longitud de lectura más corta de lo esperado

Observaciones Posibles causas 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 y preparación de la biblioteca.

1. Consulte la sección de buenas prácticas 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. DNA gel2 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

Observaciones Posibles causas Comentarios y acciones recomendadas
Gran proporción de poros no disponibles (se muestran en azul en el panel de canales y en el gráfico de actividad de poros)

image2022-3-25 10-43-25 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". Si la porción poros no disponibles se mantiene elevada o aumenta:

1. Realizar un purgado 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 (1)

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.

Fluctuación de la temperatura

Observaciones Posibles causas 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

Observaciones Posibles causas Comentarios y acciones recomendadas
MinKNOW muestra el mensaje "Error al intentar alcanzar la temperatura deseada" El dispositivo ha sido colocado en un lugar con 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 dispone de 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 continúa. Secuenciar a una temperatura incorrecta puede provocar disminuciones en el rendimiento y generar índices de calidad Qscore menores. Corrija la ubicación del dispositivo, procurando 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.

Last updated: 1/22/2025

Document options

PromethION