Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109)

Oxford Nanopore Technologies
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MinION: Protocol

Ligation sequencing gDNA - Cas9 enrichment (SQK-CS9109) V CAS_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

概览

  • 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

重要

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
重要

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

材料
  • 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)
耗材
  • 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)
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml 薄壁PCR管
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
仪器
  • 迷你离心机
  • Magnetic rack
  • 涡旋混匀仪
  • 热循环仪
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头
  • 盛有冰的冰桶
  • 计时器
可选仪器
  • Agilent生物分析仪（或等效仪器）
  • Qubit荧光计 （或用于质控检测的等效仪器）
  • Eppendorf 5424 离心机（或等效器材）

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
重要

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.

提示

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. 计算机要求及软件

MinION Mk1B的IT配置要求

请为MinION Mk1B配备一台高规格的计算机或笔记本电脑，以适配数据采集的速度。您可以在MinION Mk1B的IT配置要求文件中了解更多。

MinION Mk1C的IT配置要求

MinION Mk1C是一款集计算功能和触控屏幕于一体的便携式测序分析仪，它无需依赖任何额外设备，即可生成并分析纳米孔测序数据。您可以在 MinION Mk1C的IT配置要求文件中了解更多。

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.

测序芯片质检

我们强烈建议您在开始测序实验前，对测序芯片的活性纳米孔数进行质检。质检需在您收到MinION /GridION /PremethION测序芯片12周之内进行，或者在您收到Flongle测序芯片四周内进行。Oxford Nanopore Technologies会对活性孔数量少于以下标准的芯片进行替换** ：

测序芯片 芯片上的活性孔数确保不少于
Flongle 测序芯片 50
MinION/GridION 测序芯片 800
PromethION 测序芯片 5000

** 请注意：自收到之日起，芯片须一直贮存于Oxford Nanopore Technologies推荐的条件下。且质检结果须在质检后的两天内递交给我们。请您按照 测序芯片质检文档中的说明进行芯片质检。

4. Preparing the Cas9 ribonucleoprotein complexes (RNPs)

耗材
  • 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)
  • 0.2 ml薄壁PCR管
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
仪器
  • 热循环仪
  • Ice bucket with wet ice
  • P200 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头
重要

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

重要

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.

重要

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.

提示

crRNA and ribonucleoproteins (RNPs)

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

重要

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.

提示

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.

提示

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

5. Dephosphorylating genomic DNA

材料
  • 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
  • Phosphatase (PHOS)
耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • 0.2 ml薄壁PCR管
仪器
  • 热循环仪
  • P100 移液枪和枪头
  • P10 移液枪和枪头

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

重要

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

材料
  • 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)
耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml薄壁PCR管
仪器
  • 热循环仪
  • Ice bucket with wet ice
  • 涡旋混匀仪
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头

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.

重要

*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

材料
  • 连接测序试剂盒内的连接缓冲液（LNB）
  • Adapter Mix (AMX)
  • T4 Ligase (LIG)
耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
仪器
  • Ice bucket with wet ice
  • 涡旋混匀仪
  • P1000 移液枪和枪头
  • P100 pipette
  • P20 移液枪和枪头

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.

重要

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

材料
  • 长片段缓冲液（LFB）
  • 短片段缓冲液（SFB）
  • Oxford Nanopore测序试剂盒中的洗脱缓冲液（EB）
  • SPRI Dilution Buffer (SDB)
耗材
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P20 移液枪和枪头
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • Eppendorf 5424 离心机（或等效器材）

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

重要

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.

将样品瞬时离心，并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动，用移液枪吸去上清液。

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
步骤结束

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

可选操作

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.

提示

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

材料
  • Flow Cell Priming Kit (EXP-FLP002)
  • Loading Beads (LB)
  • Sequencing Buffer (SQB)
耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 无核酸酶水（如ThermoFisher，AM9937）
仪器
  • MinION device
  • MinION 及GridION 测序芯片遮光片
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • 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

可选操作

为文库上样前，完成测序芯片检测，查看可用孔数目。

如此前已对测序芯片进行过质检，则此步骤可省略。

更多信息，请查看MinKNOW实验手册的 测序芯片质检 部分。

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

Flow Cell Loading Diagrams Step 2

重要

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将预处理孔打开后，检查孔周围是否有小气泡。请按照以下方法，从孔中排出少量液体以清除气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入预处理孔中。
  3. 反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。
    __请注意：__ 肉眼检查，确保从预处理孔到传感器阵列的缓冲液连续且无气泡。

中文-测序芯片预处理上样3

通过预处理孔向芯片中加入800µl预处理液，避免引入气泡。等待5分钟。在此期间，请按照以下步骤准备用于上样的DNA文库。

中文-测序芯片预处理上样4

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

重要

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.

完成测序芯片的预处理：

  1. 轻轻地翻起SpotON上样孔盖，使SpotON上样孔显露出来。 中文-测序芯片预处理上样5
  2. 通过预处理孔（而 非 SpotON加样孔）向芯片中加入200µl预处理液，避免引入气泡。 中文-测序芯片预处理上样6

临上样前，用移液枪轻轻吹打混匀制备好的文库。

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

轻轻合上SpotON加样孔孔盖，确保塞头塞入加样孔内。逆时针转动预处理孔孔盖，盖上预处理孔。

中文-测序芯片预处理上样8

中文-测序芯片预处理上样9

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

按下述步骤安装测序芯片遮光片：

  1. 小心将遮光片的前沿（平端）与金属固定夹的边沿对齐。 请注意： 请勿将遮光片强行压到固定夹下方。

  2. 将遮光片轻轻盖在测序芯片上。遮光片的SpotON加样孔孔盖缺口应与芯片上的SpotON加样孔孔盖接合，遮盖住整个测序芯片的前部。

MinION加装遮光片

注意

MinION测序芯片的遮光片并非固定在测序芯片上，因此当为芯片加装遮光片后，请小心操作。

步骤结束

小心合上测序设备上盖并在MinKNOW上设置测序实验。

10. Data acquisition and basecalling

纳米孔数据分析概览

有关纳米孔数据分析的完整概述，包括碱基识别和次级分析，请参阅 数据分析 文档。

如何开始测序

MinKNOW软件负责仪器控制，数据采集和实时碱基识别。如您已在计算机上安装MinKNOW，则可选择以下几种途径开展测序：

1. 使用计算机上的MinKNOW进行实时数据采集和碱基识别

请按照 MinKNOW 实验指南 的说明：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 使用GridION进行实时数据采集和碱基识别

请参照 GridION 用户手册 中的说明。

3. 使用MinION Mk1C测序仪进行实时数据采集和碱基识别

请参照 MinION Mk1C 使用指南中的说明。

4. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 使用指南 或 PromethION 2 Solo 使用指南中的说明。

5. 使用计算机上的MinKNOW进行数据采集，过后再用NinKNOW进行线下碱基识别

请按照 MinKNOW 实验指南 中的说明：从“开始测序”部分起，到“MinKNOW运行结束”部分止。 当您设置实验参数时，请将 碱基识别 选项设为“关”。 测序实验结束后，请按照 MinKNOW 实验指南的本地分析 部分操作。

重要

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. 测序芯片的重复利用及回收

材料
  • 测序芯片清洗剂盒（EXP-WSH004）

完成测序实验后，如您希望再次使用测序芯片，请按照测序芯片清洗试剂盒的说明进行操作，并将清洗后的芯片置于2-8℃保存。

您可在纳米孔社区获取 测序芯片清洗试剂盒实验指南。

提示

我们建议您在停止测序实验后尽快清洗测序芯片。如若无法实现，请将芯片留在测序设备上，于下一日清洗。

请按照“回收程序”清洗好芯片，以便送回Oxford Nanopore。

您可在 此处找到回收测序芯片的说明。

请注意： 在将测序芯片寄回之前，请使用去离子水对每张芯片进行冲洗。

重要

如果您遇到问题或对测序实验有疑问，请参阅本实验指南在线版本中的“疑难解答指南”一节。

13. DNA/RNA提取和文库制备过程中可能出现的问题

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

低质量样本

现象 可能原因 措施及备注
低纯度DNA（Nanodrop测定的DNA吸光度比值260/280<1.8，260/230 <2.0-2.2） 用户所使用的DNA提取方法未能达到所需纯度 您可在 污染物专题技术文档 中查看污染物对后续文库制备和测序实验的影响。请尝试其它不会导致污染物残留的 提取方法。

请考虑将样品再次用磁珠纯化。
RNA完整度低（RNA完整值（RIN）<9.5，或rRNA在电泳凝胶上的条带呈弥散状） RNA在提取过程中降解 请尝试其它 RNA 提取方法。您可在 RNA完整值专题技术文档 中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅 DNA/RNA 操作 页面。
RNA的片段长度短于预期 RNA在提取过程中降解 请尝试其它 RNA 提取方法。 您可在 RNA完整值专题技术文档中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅DNA/RNA 操作 页面。

我们建议用户在无RNA酶污染的环境中操作，并确保实验设备没有受RNA酶污染.

经AMPure磁珠纯化后的DNA回收率低

现象 可能原因 措施及备注
低回收率 AMPure磁珠量与样品量的比例低于预期，导致DNA因未被捕获而丢失 1. AMPure磁珠的沉降速度很快。因此临加入磁珠至样品前，请确保将磁珠重悬充分混匀。

2. 当AMPure磁珠量与样品量的比值低于0.4:1时，所有的DNA片段都会在纯化过程中丢失。
低回收率 DNA片段短于预期 AMPure磁珠量与样品量的比值越低，针对短片段的筛选就越严格。每次实验时，请先使用琼脂糖凝胶（或其他凝胶电泳方法）确定起始DNA的长度，并据此计算出合适的AMPure磁珠用量。 SPRI cleanup
末端修复后的DNA回收率低 清洗步骤所用乙醇的浓度低于70% 当乙醇浓度低于70%时，DNA会从磁珠上洗脱下来。请确保使用正确浓度的乙醇。

14. Issues during the sequencing run

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

Mux扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数

现象 可能原因 措施及备注
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 纳米孔阵列中引入了气泡 在对通过质控的芯片进行预处理之前，请务必排出预处理孔附近的气泡。否则，气泡会进入纳米孔阵列对其造成不可逆转地损害。 视频中演示了避免引入气泡的最佳操作方法。
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 测序芯片没有正确插入测序仪 停止测序，将芯片从测序仪中取出，再重新插入测序仪内。请确保测序芯片被牢固地嵌入测序仪中，且达到目标温度。如用户使用的是GridION/PromethION测序仪，也可尝试将芯片插入仪器的其它位置进行测序。
inKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 文库中残留的污染物对纳米孔造成损害或堵塞 在测序芯片质检阶段，我们用芯片储存缓冲液中的质控DNA分子来评估活性纳米孔的数量。而在测序开始时，我们使用DNA文库本身来评估活性纳米孔的数量。因此，活性纳米孔的数量在这两次评估中会有约10%的浮动。

如测序开始时报告的孔数明显降低，则可能是由于文库中的污染物对膜结构造成了损坏或将纳米孔堵塞。用户可能需要使用其它的DNA/RNA提取或纯化方法，以提高起始核酸的纯度。您可在 污染物专题技术文档中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的 提取方法 。

MinKNOW脚本失败

现象 可能原因 措施及备注
MinKNOW显示 "Script failed”（脚本失败）
重启计算机及MinKNOW。如问题仍未得到解决，请收集 MinKNOW 日志文件 并联系我们的技术支持。 如您没有其他可用的测序设备，我们建议您先将装有文库的测序芯片置于4°C 储存，并联系我们的技术支持团队获取进一步储存上的建议。

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.

读长短于预期

现象 可能原因 措施及备注
读长短于预期 DNA样本降解 读长反映了起始DNA片段的长度。起始DNA在提取和文库制备过程中均有可能被打断。

1. 1. 请查阅纳米孔社区中的 提取方法 以获得最佳DNA提取方案。

2. 在进行文库制备之前，请先跑电泳，查看起始DNA片段的长度分布。DNA gel2 在上图中，样本1为高分子量DNA，而样本2为降解样本。

3. 在制备文库的过程中，请避免使用吹打或/和涡旋振荡的方式来混合试剂。轻弹或上下颠倒离心管即可。

大量纳米孔处于不可用状态

现象 可能原因 Comments and actions
大量纳米孔处于不可用状态 (在通道面板和纳米孔活动状态图上以蓝色表示)

image2022-3-25 10-43-25 上方的纳米孔活动状态图显示：状态为不可用的纳米孔的比例随着测序进程而不断增加。		 样本中含有污染物 使用MinKNOW中的“Unblocking”（疏通）功能，可对一些污染物进行清除。 如疏通成功，纳米孔的状态会变为"测序孔". 若疏通后，状态为不可用的纳米孔的比例仍然很高甚至增加：

1. 用户可使用 测序芯片冲洗试剂盒（EXP-WSH004）进行核酸酶冲洗 can be performed, 操作，或
2. 使用PCR扩增目标片段，以稀释可能导致问题的污染物。

大量纳米孔处于失活状态

现象 可能原因 措施及备注
大量纳米孔处于失活状态（在通道面板和纳米孔活动状态图上以浅蓝色表示。膜结构或纳米孔遭受不可逆转地损伤） 测序芯片中引入了气泡 在芯片预处理和文库上样过程中引入的气泡会对纳米孔带来不可逆转地损害。请观看 测序芯片的预处理及上样 视频了解最佳操作方法。
大量纳米孔处于失活/不可用状态 文库中存在与DNA共纯化的化合物 与植物基因组DNA相关的多糖通常能与DNA一同纯化出来。

1. 请参考 植物叶片DNA提取方法。
2. 使用QIAGEN PowerClean Pro试剂盒进行纯化。
3. 利用QIAGEN REPLI-g试剂盒对原始gDNA样本进行全基因组扩增。
大量纳米孔处于失活/不可用状态 样本中含有污染物 您可在 污染物专题技术文档 中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的提取方法。

运行过程中过孔速度和数据质量（Q值）降低

现象 可能原因 措施及备注
运行过程中过孔速度和数据质量（Q值）降低 对试剂盒9系列试剂（如SQK-LSK109），当测序芯片的上样量过多时（请参阅相应实验指南获取推荐文库用量），能量消耗通常会加快。 请按照MinKNOW 实验指南中的说明为测序芯片补充能量。请在后续实验中减少测序芯片的上样量。

温度波动

现象 可能原因 措施及备注
温度波动 测序芯片和仪器接触不良 检查芯片背面的金属板是否有热垫覆盖。重新插入测序芯片，用力向下按压，以确保芯片的连接器引脚与测序仪牢固接触。如问题仍未得到解决，请联系我们的技术支持。

未能达到目标温度

现象 可能原因 措施及备注
MinKNOW显示“未能达到目标温度” 测序仪所处环境低于标准室温，或通风不良（以致芯片过热） MinKNOW会限定测序芯片达到目标温度的时间。当超过限定时间后，系统会显示出错信息，但测序实验仍会继续。值得注意的是，在错误温度下测序可能会导致通量和数据质量（Q值）降低。请调整测序仪的摆放位置，确保其置于室温下、通风良好的环境中后，再在MinKNOW中继续实验。有关MinION MK1B温度控制的更多信息，请参考此 FAQ （常见问题）文档。

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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