Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109)

Oxford Nanopore Technologies
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Protocol

Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109) VCAS_9106_v109_revH_16Sep2020

  • We advise all customers to read and consider the information in the "Targeted, amplification-free DNA sequencing using CRISPR/Cas" info sheet before starting this protocol
  • This protocol uses genomic DNA
  • Targeted cutting around specific genomic Regions of Interest (ROI) to achieve enrichment
  • Library preparation time is ~110 minutes
  • Fragment lengths are determined by the cut spacing, and not fragmentation
  • No PCR is required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

FOR RESEARCH USE ONLY

Overview

  • We advise all customers to read and consider the information in the "Targeted, amplification-free DNA sequencing using CRISPR/Cas" info sheet before starting this protocol
  • This protocol uses genomic DNA
  • Targeted cutting around specific genomic Regions of Interest (ROI) to achieve enrichment
  • Library preparation time is ~110 minutes
  • Fragment lengths are determined by the cut spacing, and not fragmentation
  • No PCR is required

For Research Use Only

This is a Legacy product This kit is soon to be discontinued. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

Document version: CAS_9106_v109_revH_16Sep2020

1. Overview of the protocol

Features of using Cas9 targeted sequencing

We recommend CRISPR/Cas targeted sequencing if the user:

  • Wishes to sequence multiple human gene targets (up to 100 in a single panel) to high coverage (>100x) on a single MinION Mk1B flow cell
  • Wishes to sequence up to a 50 kb Region of Interest (ROI), using up to 100 target sites, in a single assay*
  • Has 1-10 µg of available gDNA
  • Wishes to gain insight into methylation patterns or other modified bases
  • Has gene targets that are highly repetitive, or wishes to evaluate the number of repeats in an expansion, where traditional amplification methods or sequencing-by-synthesis methods could yield a biased result
  • Wishes to sequence long gene targets in a single pass that are not amenable to long-range PCR (> 30 kb)
  • Optionally wishes to run multiple barcoded samples on a single flow cell.

* Note: This is known as ‘tiling’ an ROI.

Excision/single cut vs tiling approach

This protocol can be used for any probe design method (details of which can be found in the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet). The recommended ‘excision approach’ and ‘single cut and read out’ method can follow the main flow of this protocol (shown in Figure 1). The ‘tiling’ approach requires the alterations to the protocol described in the Important (orange) boxes in the library preparation section. The main difference with the tiling approach is that both pools of probes need to be prepared separately (RNP complex formation, cleavage and dA-tailing and adapter ligation performed in separate tubes) then pooled during the final AMPure XP bead purification (shown in Figure 2).

Single pool Figure 1. Cas9 targeted sequencing protocol using the 'excision approach' or 'single cut and read out'.

tiling Figure 2. Cas9 targeted sequencing protocol using the 'tiling' approach

IMPORTANTE

Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet

For more details about the Cas9 targeted sequencing approach, how to design probes, and general expectations and guidance, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet. We strongly recommend that you read it before proceeding with your targeted sequencing experiments.

Introduction to the CRISPR/Cas protocol

This protocol describes how to carry out sequencing of genomic DNA using the Cas9 Sequencing Kit (SQK-CS9109) with enrichment of specific genomic regions using CRISPR/Cas.

For users with no previous nanopore sequencing experience, we recommend that a Lambda control experiment is completed first to become familiar with the technology.

Prepare for your experiment You will need to:

  • Extract your DNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
  • Ensure you have your sequencing kit, the correct equipment and third-party reagents
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Library preparation Figure 1. shows and explains the biochemical steps used to prepare your barcoded DNA library using a Cas9 Sequencing Kit (SQK-CS9109), plus several third-party reagents.

Cas9 sequencing workflow

Figure 1. Cas-mediated PCR-free enrichment library preparation for sequencing.

  1. After DNA extraction, 5’ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands.
  2. Cas9 ribonucleoprotein particles (RNPs), with bound crRNA and tracrRNA, are added to the genomic DNA, then bind and cleave the Region of Interest (ROI).
  3. dsDNA cleavage by Cas9 reveals blunt ends with ligatable 5’ phosphates.
  4. All of the DNA in the samples are dA-tailed, which prepares the blunt ends for barcode ligation.
  5. Sequencing adapters are ligated primarily to Cas9 cut sides, which are both 3’ dA-tailed and 5’ phosphorylated. The library preparation is cleaned up to remove excess adapters using AMPure XP beads and resuspended in Sequencing Buffer. Non-target molecules are not removed. The subsequent library preparation is added to the flow cell for sequencing.

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
  • Start the EPI2ME software and select a workflow for further analysis (this step is optional)

Enrichment experiment steps and associated instructions

Step Instructions
1. Extract and prepare DNA Extraction methods
2. Design probes Targeted, amplification-free DNA sequencing using CRISPR/Cas - Probe design
3. QC input DNA Input DNA/RNA QC
4. Perform enrichment, and prepare sequencing library Cas Sequencing Kit protocol
5. Sequence on device Cas Sequencing Kit protocol
6. Take basecalled FASTQ files into analysis pipeline Targeted, amplification-free DNA sequencing using CRISPR/Cas - Evaluation of read-mapping characteristics from a Cas9 targeted sequencing experiment
7. Assess success of experiment and feed back into probe design and quality of input
IMPORTANTE

Compatibility of this protocol

This protocol should only be used in combination with:

  • Cas9 Sequencing Kit (SQK-CS9109)
  • R9.4.1 (FLO-MIN106) flow cells
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

Material
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Cas9 Sequencing Kit (SQK-CS9109)
  • Flow Cell Priming Kit (EXP-FLP002)
Consumibles
  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
  • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Tubos de PCR de pared fina (0,2 ml)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
Instrumental
  • Microcentrífuga
  • Magnetic rack
  • Mezclador vórtex
  • Termociclador
  • 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
  • Bioanalizador Agilent (o equivalente)
  • Qubit fluorometer (or equivalent for QC check)
  • Centrifuga Eppendorf 5424 (o equivalente)

For this protocol, you will need 1–10 µg or 0.1–2 pmol high molecular weight genomic DNA (5 µg recommended).

Cas9 Sequencing Kit contents (SQK-CS9109)

cs9109 v1

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Adapter Mix AMX Green 2 40
Ligation Buffer LNB Clear 2 200
Elution Buffer EB Black 1 200
Sequencing Buffer SQB Red 2 300
L Fragment Buffer LFB Orange 2 1,800
S Fragment Buffer SFB Grey 2 1,800
Loading Beads LB Pink 1 360
Phosphatase PHOS Brown tube, yellow label 1 50
TAQ Polymerase TAQ Brown tube, green label 1 15
SPRI Dilution Buffer SDB Brown tube, red label 1 1,200
T4 DNA Ligase LIG Brown tube, blue label 1 140
dATP dATP Brown tube, grey label 1 15
Reaction Buffer RB Brown tube, orange label 1 180

Flow Cell Priming Kit contents (EXP-FLP002)

FLP

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Flush Buffer FB Blue 6 1,170
Flush Tether FLT Purple 1 200
IMPORTANTE

Genomic DNA and its quality

Unsheared, high-molecular weight DNA, as isolated e.g., using the Qiagen Genomic DNA Kit, at ≥210 ng/µl by Qubit, and stored in TE (pH 8.0) or similar, or nuclease-free water.

Carryover of 1 mM EDTA from TE will not significantly affect this protocol; however, care should be taken to reduce other contaminants, such as detergents, phenol, chloroform, and salts.

CONSEJO

Wide-bore tips

Use wide-bore tips (or regular pipette tips with the narrow ends cut off) where possible to minimise shearing of long DNA.

Sourcing crRNA and tracrRNA

We recommend using synthetic crRNA and tracrRNA from IDT, which are of sufficient purity and carry modifications that confer stability and nuclease resistance. For this reason we caution against using single-guide RNAs (sgRNAs).

  • S. pyogenes Cas9 Alt-R™ crRNAs
  • S. pyogenes Cas9 tracrRNA (e.g. IDT Alt-R™, Cat # 1072532, 1072533 or 1072534)

Individual crRNAs and tracrRNA should be resuspended at 100 µM each in TE, pH 7.5, aliquoted to avoid freeze-thawing, and stored at –20° C for up to two weeks or –80° C if stored long-term. The crRNAs/tracrRNAs can be freeze-thawed a maximum of five times.

crRNAs may be pooled to make panels for generating multiple cuts in a single reaction. To pool crRNAs, we recommend dispensing equal volumes of each crRNA (up to 100 crRNAs, each at 100 µM) into a separate 1.5 ml Eppendorf DNA LoBind tube to make an equimolar crRNA mix.

We strongly recommend TE at pH 7.5, rather than pH 8.0, for the long-term stability of RNA oligos in storage.

3. Computer requirements and software

Requisitos informáticos para el MinION Mk1B

Para secuenciar con el MinION Mk1B es necesario tener un ordenador o portátil de alto rendimiento, que pueda soportar la velocidad de adquisición de datos. Encontrará más información en el documento MinION Mk1B IT Requirements.

Requisitos informáticos para el MinION Mk1C

El MinION Mk1C contiene ordenador y pantalla integrados, lo que elimina la dependencia de cualquier accesorio para generar y analizar datos de nanoporos. Encontrará más información en el documento MinION Mk1C IT Requirements.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform protocol.

Verificar la celda de flujo

Antes de empezar el experimento de secuenciación, recomendamos verificar el número de poros disponibles, presentes en la celda de flujo. La comprobación deberá realizarse en los primeros tres meses desde su adquisición, si se trata de celdas de flujo MinION, GridION o PromethION, y en las primeras cuatro semanas tras la compra de celdas de flujo Flongle. Oxford Nanopore Technologies sustituirá cualquier celda de flujo con un número de poros inferior al indicado en la tabla siguiente, siempre y cuando el resultado se notifique dentro de los dos días siguientes a la comprobación y se hayan seguido las instrucciones de almacenamiento. Para verificar la celda de flujo, siga las instrucciones del documento Flow Cell Check.

Celda de flujo Número mínimo de poros activos cubierto por la garantía
Flongle 50
MinION/GridION 800
PromethION 5000

4. Preparing the Cas9 ribonucleoprotein complexes (RNPs)

Consumibles
  • S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
  • Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
  • S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
  • Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
  • Tubos de PCR de pared fina (0,2 ml)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
Instrumental
  • Termociclador
  • Ice bucket with wet ice
  • Pipeta y puntas P200
  • Pipeta y puntas P10
  • Pipeta y puntas P2
IMPORTANTE

Here, the Cas9 is loaded with crRNA and tracrRNA to form ribonucleoprotein complexes (RNPs) in preparation for the cleavage reaction.

IMPORTANTE

If using a 'tiling' approach

The formula below prepares a pool of RNPs for making multiple excisions in a single reaction. If using a tiling approach for probe design (a method for designing probes in two separate overlapping pools to cover a target region >20 kb), prepare 2x RNP complexes, one for each pool of crRNA probes. For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

IMPORTANTE

RNP complex stability

Upon receipt, we recommend aliquoting individual probes or pools of crRNAs for storage, to minimise freeze-thawing.

Panels of RNPs may be formed ahead of time and stored at 4°C for up to a week, or at -80°C for up to a month with no discernible loss of activity. However, we recommend making a fresh RNP complex for every experiment if possible.

CONSEJO

crRNA and ribonucleoproteins (RNPs)

The following protocol applies to a single crRNA. For panels of multiple crRNAs, see “Notes on multiple crRNAs” below.

IMPORTANTE

Notes on multiple crRNAs

If you have validated a set of crRNA probes that are always run together, we recommend pooling all crRNA probes and then aliquoting that pool and freezing each aliquot at -80°C. Each time you run a Cas9 experiment and need to make an RNP complex, take out a crRNA pool aliquot and combine with the tracrRNA.

Pre-heat a thermal cycler to 95ºC.

Thaw an aliquot of Reaction Buffer (RB), mix by vortexing, and place on ice.

In an 1.5 ml Eppendorf DNA LoBind tube, pool the crRNA probes for each cleavage reaction by combining equal volumes of each crRNA probe, resuspended at 100 µM in TE (pH 7.5).

  • A single crRNA or many crRNA probes (up to ~100) may be used in a single cleavage reaction.
  • The crRNA probes may also be pre-mixed as an off-catalogue request from IDT.
  • For example, probes for the HTT gene, found here, can be used as an individual experiment or in addition to other probes as an in-run control.
  • Unused crRNA probe mix may be stored at -80ºC and minimal freeze thaw recommended.

Anneal the pooled crRNAs with tracrRNA in Duplex Buffer by assembling the following in a 0.2 ml thin-walled PCR tube, as follows:

Reagent Volume
Duplex buffer 8 µl
crRNA pool (100 µM, equimolar) 1 µl
tracrRNA (100 µM) 1 µl
Total 10 µl

Mix well by pipetting and spin down.

Using a thermal cycler heat the above reaction mix at 95ºC for 5 mins, then remove the tube from the thermal cycler and allow it to cool to room temperature, then spin down the tube to collect any liquid in the bottom of the tube.

  • Storage and reuse of the annealed mix is not recommended.

To form Cas9 RNPs, assemble the components in the table in an 1.5 ml Eppendorf DNA LoBind tube; this will form the annealed crRNA•tracrRNA, through pooling in the stated order:

Reagent Volume
Nuclease-free water 79.2 µl
Reaction Buffer (RB) 10 µl
Annealed crRNA•tracrRNA pool (10 µM) 10 µl (Step 4, above)
HiFi Cas9 (62 µM) 0.8 µl
Total 100 µl

Note: Refer to the tip below for scaling-down this ternary RNP mix.

Mix thoroughly by flicking the tube.

CONSEJO

The above step yields an excess amount of RNPs, but 10 µl are carried forwards for each reaction into the next target cleavage step. Any excess RNPs may be stored at 4ºC for up to a week. The reaction may be scaled, as shown in the below table.

Number of reactions 3 5 10
Components Volume (µl) Volume (µl) Volume (µl)
Annealed crRNA•tracrRNA pool (10 µM) (Step 1) 3 5 10
Reaction Buffer (RB) 3 5 10
Nuclease-free water 23.7 39.6 79.2
HiFi Cas9 (62 µM) 0.3 0.4 0.8
Total 30 50 100

Form the RNPs by incubating the tube at room temperature for 30 minutes, then return the RNPs on ice until required.

CONSEJO

Proceed to the next step (gDNA dephosphorylation) during the 30 min RNP incubation step.

5. Dephosphorylating genomic DNA

Material
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Phosphatase (PHOS)
Consumibles
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Tubos de PCR de pared fina (0,2 ml)
Instrumental
  • Termociclador
  • Pipeta y puntas P100
  • Pipeta y puntas P10

This step reduces background reads by removing 5’ phosphates from non-target DNA ends.

IMPORTANTE

If using a 'tiling' approach

If using a tiling approach for probe design (a method for designing probes in two separate overlapping pools to cover a target region >20 kb), and have just produced 2x separate RNP complexes, users need to perform the dephosphorylation, Cas9 cleavage and adapter ligation step twice (one reaction per pool of probes). For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

Prepare the DNA in nuclease-free water

  • Transfer 1-10 μg (with 5 μg recommended) genomic DNA into a 0.2 ml thin-walled PCR tubes
  • Adjust to 24 µl with nuclease-free water
  • Mix thoroughly by flicking the tube avoiding unwanted shearing
  • Spin down briefly in a microfuge

Mix the Phosphatase (PHOS) in the tube by pipetting up and down. Ensure that it is at room temperature before use.

Assemble the following components in a clean 0.2 ml thin-walled PCR tube:

Reagent Volume
Reaction Buffer (RB) 3 µl
HMW genomic DNA (at ≥ 210 ng/µl)* 24 µl
Total 27 µl
  • Note: For an initial test, we recommend 5 µg genomic DNA input. Preparing input DNA step yields ~100-2000x target coverage. Target coverage scales linearly with input amount, so the input amount may be reduced accordingly if lower throughput is acceptable.

Ensure the components are thoroughly mixed by pipetting, and spin down.

Add 3 µl of PHOS to the tube.

Mix gently by flicking the tube, and spin down.

Using a thermal cycler, incubate at 37ºC for 10 minutes, 80ºC for 2 minutes then hold at 20ºC (room temperature).

6. Cleaving and dA-tailing target DNA

Material
  • 5 µg high molecular weight dephosphorylated genomic DNA (recommended); 1 - 10 µg (or 0.1-2 pmol) can be used accordingly.
  • crRNA-tracrRNA-Cas9 ribonucleoprotein complexes (RNPs)
  • Taq Polymerase (TAQ)
  • dATP (dATP)
Consumibles
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Tubos de PCR de pared fina (0,2 ml)
Instrumental
  • Termociclador
  • Ice bucket with wet ice
  • Mezclador vórtex
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • Pipeta y puntas P2

In this step, Cas9 RNPs (see 'Preparing the Cas9 ribonucleoprotein complexes') and Taq polymerase are added to the dephosphorylated genomic DNA sample.

This process cleaves target and dA-tails all available DNA ends in one step, activating the Cas9 cut sites for ligation.

Thaw the dATP tube, vortex to mix thoroughly and place on ice.

Spin down and place the tube of Taq Polymerase (TAQ) on ice.

To the PCR tube containing 30 µl dephosphorylated DNA sample, add:

Reagent Volume
Dephosphorylated genomic DNA sample 30 µl
Cas9 RNPs 10 µl
dATP 1 µl
Taq Polymerase (TAQ) 1 µl
Total 42 µl

Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler.

Using the thermal cycler, incubate at 37ºC for 15-60 minutes*, then 72ºC for 5 minutes and hold at 4ºC or return to tube to ice.

IMPORTANTE

*The Cas9 enzyme is active at 37ºC, and denatured at 72ºC. We recommend a 15 minute cut time by default. Longer 37ºC incubations may increase the amount of off-target reads without increasing the yield of on-target reads, while shorter incubations may result in incomplete target cleavage. However, some regions may benefit from a longer incubation at 37ºC.

7. Adapter ligation

Material
  • Ligation Buffer (LNB) (tampón de ligación) del kit Ligation Sequencing Kit
  • Adapter Mix (AMX)
  • T4 Ligase (LIG)
Consumibles
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
Instrumental
  • Ice bucket with wet ice
  • Mezclador vórtex
  • Pipeta y puntas P1000
  • P100 pipette
  • Pipeta y puntas P20

Here, AMX adapters from the Cas Sequencing Kit (SQK-CS9109) are ligated to the ends generated by Cas9 cleavage.

Thaw the Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

Carefully transfer the contents of the 0.2 ml thin-walled PCR tube to a fresh 1.5 ml Eppendorf DNA LoBind Tube using a wide-bore pipette tip.

Thaw an aliquot of Adapter Mix (AMX), mix by flicking the tube, pulse-spin to collect the liquid in the bottom of the vial, then return the vial to ice.

Bring the AMPure XP beads to room temperature.

Assemble the following at room temperature in a separate 1.5 ml Eppendorf DNA LoBind Tube, adding Adapter Mix (AMX) last, before you are ready to begin the ligation:

Reagent Volume
Ligation Buffer (LNB) 20 µl
Nuclease-free water 3 µl
T4 Ligase (LIG) 10 µl
Adapter Mix (AMX)* 5 µl
Total 38 µl

Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed.

IMPORTANT: Add 20 µl of the adapter ligation mix to the cleaved and dA-tailed sample. Mix gently by flicking the tube. Do not centrifuge the sample at this stage. Immediately after mixing, add the remainder of the adapter ligation mix to the cleaved and dA-tailed sample, to yield an 80 µl ligation mix.

Ensure the components are thoroughly mixed by pipetting, and spin down.

Incubate the reaction for 10 minutes at room temperature.

IMPORTANTE

DNA precipitation

A white precipitate may form upon addition of the adapter ligation mix to the dA-tailed DNA. Adding the ligation mixture in two parts helps to reduce precipitation. However, the presence of a precipitate does not necessarily indicate failure of ligation of the sequencing adapter to target molecule ends.

8. AMPure XP bead purification

Material
  • Long Fragment Buffer (LFB) (tampón para fragmentos largos)
  • Short Fragment Buffer (SFB) (tampón para fragmentos cortos)
  • Elution Buffer (EB) (tampón de elución) del kit de Oxford Nanopore
  • SPRI Dilution Buffer (SDB)
Consumibles
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • Tubos de 1,5 ml Eppendorf DNA LoBind
Instrumental
  • Pipeta y puntas P1000
  • Pipeta y puntas P200
  • Pipeta y puntas P20
  • Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
  • Eppendorf 5424 centrifuge (or equivalent)

This step removes excess unligated adapters and other short DNA fragments, and concentrates and buffer-exchanges the library in preparation for sequencing.

IMPORTANTE

If using a 'tiling' approach

Complete steps 1 and either 2 or 3 depending on the DNA fragment lengths you wish to retain. Then pool the samples together into a single tube, before performing steps 4, 5, and 6 with the modified volumes for your pooled sample. This includes 1 volume (160 µl) of SPRI Dilution Buffer (SDB) and 0.3X (96 µl) of AMPure XP beads. For more information about tiling, please refer to the Targeted, amplification-free DNA sequencing using CRISPR/Cas info sheet.

Thaw the Elution Buffer (EB) and SPRI Dilution Buffer (SDB) at room temperature, mix by vortexing, spin down and place on ice.

To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.

To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.

Add 1 volume (80 µl) of the SPRI Dilution Buffer (SDB) to the ligation mix. Mix gently by flicking the tube.

Resuspend the AMPure XP beads by vortexing.

Add 0.3x volume (48 µl) of AMPure XP Beads to the ligation sample. The volume of beads is calculated based on the volume after the addition of SDB. Mix gently by inversion. If any sample ends up in the lid, spin down the tube very gently, keeping the beads suspended in liquid.

Incubate the sample for 10 minutes at room temperature. Do not agitate or pipette the sample.

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 either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB), depending on the size of your target molecule. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow 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 pellet in 13 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature.

Note: For targets >30 kb, we recommend increasing the elution time to 30 minutes.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 12 µl of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube.

  • Dispose of the pelleted beads
FIN DEL PROCESO

The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.

MEDIDA OPCIONAL

If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the libraries onto flow cells.

CONSEJO

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, reloading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes. For further information, please refer to the DNA library stability Know-How document.

9. Priming and loading the SpotON flow cell

Material
  • Flow Cell Priming Kit (EXP-FLP002)
  • Loading Beads (LB)
  • Sequencing Buffer (SQB)
Consumibles
  • Tubos de 1,5 ml Eppendorf DNA LoBind
  • Agua sin nucleasas (p. ej., ThermoFisher AM9937)
Instrumental
  • MinION device
  • Pantalla protectora para celdas de flujo MinION
  • Pipeta y puntas P1000
  • Pipeta y puntas P100
  • Pipeta y puntas P20
  • Pipeta y puntas P10
  • SpotON Flow Cell

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down at room temperature.

To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

Open the MinION Mk1B lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 1b

MEDIDA OPCIONAL

Antes de cargar la biblioteca, verifique la celda de flujo para determinar el número de poros disponible.

Si se ha verificado con anterioridad la cantidad de poros presentes en la celda de flujo, este paso se puede omitir.

Dispone de más información en las instrucciones de comprobación de la celda de flujo, del protocolo de MinKNOW.

Slide the priming port cover clockwise to open the priming port.

Flow Cell Loading Diagrams Step 2

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón. 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.

Tras abrir el puerto de cebado, verificar si hay una burbuja de aire bajo la tapa. Retirar una pequeña cantidad de tampón para quitar las burbujas:

  1. Ajustar una pipeta P1000 a 200 μl.
  2. Introducir la punta de la pipeta en el puerto de cebado.
  3. Girar la rueda hasta que el indicador de volumen marque 220-230 μl o hasta que se pueda ver una pequeña cantidad de tampón entrar en la punta de la pipeta.

Nota: Comprobar que haya un flujo continuo de tampón circulando desde el puerto de cebado a través de la matriz de poros.

Flow Cell Loading Diagrams Step 03 V5

Cargar 800 μl de mezcla de cebado en el puerto de cebado, evitando introducir burbujas de aire. Esperar 5 minutos. Durante este tiempo, preparar la biblioteca para cargar siguiendo los pasos a continuación.

Flow Cell Loading Diagrams Step 04 V5 SPANISH

Thoroughly mix the contents of the Loading Beads (LB) by pipetting.

IMPORTANTE

The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

In a new tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer (SQB) 37.5 µl
Loading Beads (LB), mixed immediately before use 25.5 µl
DNA library 12 µl
Total 75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.

Completar el cebado de la celda de flujo:

  1. Levantar suavemente la tapa del puerto de muestra SpotON.
  2. Cargar 200 µl de mezcla de cebado en el puerto de cebado (no en el puerto de muestra SpotON), evitando introducir burbujas de aire.

Flow Cell Loading Diagrams Step 5

Flow Cell Loading Diagrams Step 06 V5 SPANISH 2

Mezclar la biblioteca pipeteando suavemente, justo antes de cargar.

Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

Flow Cell Loading Diagrams Step 07 V5

Volver a colocar con cuidado, la tapa del puerto de muestra SpotON, procurando que el tapón encaje en el agujero y cerrar el puerto de cebado.

Step 8 update - SPANISH

Flow Cell Loading Diagrams Step 9 SPANISH

IMPORTANTE

Para obtener resultados de secuenciación óptimos, instale la pantalla protectora justo después de cargar la biblioteca.

Recomendamos poner 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.

Colocar la pantalla protectora de la siguiente manera:

  1. Colocar con cuidado el borde delantero de la pantalla protectora contra el clip. Nota: No hacer fuerza sobre ella.

  2. Colocar la pantalla protectora con suavidad sobre la celda de flujo. La pieza debe asentarse alrededor de la tapa SpotON y debe cubrir por completo la sección superior de la celda de flujo.

J2264 - Light shield animation Flow Cell FAW optimised. SPANISH

ATENCIÓN

La pantalla protectora no está fijada a la celda de flujo. Una vez colocada, es necesario manipularla con cuidado.

FIN DEL PROCESO

Cerrar la tapa del dispositivo y configurar un experimento de secuenciación en MinKNOW.

10. 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.

Cómo empezar a secuenciar

El programa MinKNOW realiza el control del dispositivo de secuenciación, la adquisición de datos y la identificación de bases en tiempo real. Una vez que el usuario ha instalado MinKNOW en su ordenador, hay diferentes maneras de llevar a cabo la secuenciación:

1. Adquisición de datos e identificación de bases en tiempo real con el programa MinKNOW.

Seguir las instrucciones del protocolo de MinKNOW, desde el apartado "Starting a sequencing run" hasta el final del apartado "Completing a MinKNOW run".

2. Adquisición de datos e identificación de bases en tiempo real con el dispositivo GridION.

Seguir las instrucciones del manual de usuario de GridION.

3. Adquisición de datos e identificación de bases en tiempo real con el dispositivo MinION Mk1C.

Seguir las instrucciones del manual de usuario de MinION Mk1C.

4. Adquisición de datos e identificación de bases en tiempo real con el dispositivo PromethION.

Seguir las instrucciones de los manuales de usuario de PromethION o PromethION 2 Solo.

5. Adquisición de datos e identificación de bases posterior mediante MinKNOW.

Seguir las instrucciones del protocolo de MinKNOW, desde el apartado "Starting a sequencing run" hasta el final del apartado "Completing a MinKNOW run". Al configurar los parámetros del experimento, ajustar la pestaña Basecalling (Identificación de bases) en posición de APAGADO. Al terminar el experimento de secuenciación, seguir las instrucciones del apartado "Post-run analysis" del protocolo de MinKNOW.

IMPORTANTE

When selecting the sequencing kit in MinKNOW, please choose SQK-CAS109 instead of SQK-LSK109.

Understanding Cas enrichment

The Duty Time feature in the MinKNOW software can be used to judge the quality of your experiment. The duty time plot shows the distribution of channel states over time, grouped by time chunks, or 'buckets'. The basic view shows the five main channel states: Sequencing, Pore, Recovering, Inactive, and Unclassified. Clicking the "More" button shows a more detailed breakdown of channel states.

It is recommended to observe the duty time plot populating over the first 30 min-1 hr of the sequencing run. By this time, the channel state distribution will give an indication whether the DNA library is of a good quality, and whether the flow cell is performing well.

Note: The Duty Time plots will be noticeably different to a conventional SQK-LSK109 run. A much smaller percentage of pores will be observed as Sequencing/Strand.

If Active Channel Selection is enabled during the run, the software instantly switches to a new channel in the group if a channel is in the “Saturated” or “Multiple” state, or after ~5 minutes if a channel is “Recovering”. This feature maximises the number of channels sequencing at the start of the experiment, however this may also result in an artificially high number of "Sequencing" or "Pore" channels in the duty time plot. For this reason, we recommend referring to the Mux Scan Results plot, which shows the true distribution of channel states at the point of the most recent mux scan.

Understanding Duty Time plots during a Cas9 targeted sequencing run

As discussed above, the user should expect a lower proportion of pores in Sequencing compared to a standard SQK-LSK109 run, while the total number of available pores should be roughly consistent between a Cas9 targeted sequencing experiment and SQK-LSK109 experiment.

Throughput duty time

FLO-MIN106 Duty time plot for a Cas9 targeted sequencing experiment using a human gene. From the Duty Time plot, there is an equivalent number of active pores between a SQK-LSK109 run and Cas9 taregeted sequencing run. In a Cas9 experiment, the sequencing pore is roughly 5-15% (light green) of the total number of pores.

11. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. Bioinformatics tutorials

For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials, which are available in the Bioinformatics resource section of the Community. The tutorials take the user through installing and running pre-built analysis pipelines, which generate a report with the results. The tutorials are aimed at biologists who would like to analyse data without the help of a dedicated bioinformatician, and who are comfortable using the command line.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

3. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the Community-developed data analysis tool library. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

12. Reutilización y devoluciones de las celdas de flujo

Material
  • Flow Cell Wash Kit (EXP-WSH004) (kit de lavado de celda de flujo)

Si al terminar el experimento desea volver a usar la celda de flujo, siga las instrucciones del protocolo Flow Cell Wash Kit y guarde la celda de flujo lavada a 2-8 ⁰C.

El protocolo Flow Cell Wash Kit está disponible en la comunidad Nanopore.

CONSEJO

Una vez terminado el experimento, recomendamos lavar la celda de flujo cuanto antes. Si no es posible, se puede dejar en el dispositivo y lavar al día siguiente.

Otra posibilidad es seguir el procedimiento de devolución para lavar la celda de flujo y enviarla a Oxford Nanopore.

Aquí puede encontrar las instrucciones para devolver celdas de flujo.

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. 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. 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 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. 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

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)

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" (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 están 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 acabado el 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 de secuenciación para asegurarse de que se encuentra a temperatura ambiente y con buena ventilación; a continuación, reinicie el proceso en MinKNOW. Para obtener más información sobre el control de temperatura de MinKNOW Mk 1B, consulte la sección de preguntas frecuentes, FAQ.

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.

Last updated: 11/1/2023

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