Ligation sequencing gDNA - automated Hamilton NGS STAR 96 (SQK-LSK109-XL)

Oxford Nanopore Technologies
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PromethION: Protocol

Ligation sequencing gDNA - automated Hamilton NGS STAR 96 (SQK-LSK109-XL) V GDA_9107_v109_revM_30Oct2020

  • This protocol uses genomic DNA
  • Automation of library preparation
  • Increased reproducibility and speed
  • Reduces human error
  • High sequencing output
  • Library preparation time ~1.5 hours hands-on-time and ~3-4 hours automation time
  • Fragmentation is optional
  • No PCR required
  • Multiple samples can be prepared simultaneously
  • Compatible with R10.3 flow cells

For Research Use Only

This is a Legacy product This kit has now been discontinued and we recommend all customers upgrade to the latest chemistry for their relevant kit which is available on the Store.

FOR RESEARCH USE ONLY

概览

  • This protocol uses genomic DNA
  • Automation of library preparation
  • Increased reproducibility and speed
  • Reduces human error
  • High sequencing output
  • Library preparation time ~1.5 hours hands-on-time and ~3-4 hours automation time
  • Fragmentation is optional
  • No PCR required
  • Multiple samples can be prepared simultaneously
  • Compatible with R10.3 flow cells

For Research Use Only

This is a Legacy product This kit has now been discontinued and we recommend all customers upgrade to the latest chemistry for their relevant kit which is available on the Store.

Document version: GDA_9107_v109_revM_30Oct2020

1. Overview of the protocol

重要

This is a Legacy product

This kit has now been discontinued and we recommend all customers upgrade to the latest chemistry for their relevant kit which is available on the Store. For further information on please see the product update page.

Ligation Sequencing Kit XL features

This kit is recommended for users who:

  • want to optimise their sequencing experiment for throughput
  • would like to utilise upstream processes such as size selection, whole genome amplification, or enrich for long reads
重要

可选步骤：DNA片段化以及片段大小筛选

本实验手册不包含DNA片段化步骤。但在某些情况下，将样品片段化可能有助于您的实验。例如，当起始gDNA量较少时（100ng-500ng），将DNA片段化能扩充分子数量，从而提高通量。请参考：牛津纳米孔社区“Extraction methods”（提取方法）板块的 DNA片段化部分。

我们也提供了一些用于富集DNA样品中长片段的方法，请参考：牛津纳米孔社区“Extraction methods”(提取方法) 板块的 片段大小筛选部分。

Introduction to the automated Ligation Sequencing protocol for gDNA

This protocol describes how to carry out sequencing of a DNA sample using the Ligation Sequencing Kit XL (SQK-LSK109-XL). It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

To efficiently load multiple PromethION Flow Cells, we recommend using the Loading multiple PromethION Flow Cells protocol as a guideline.

Steps in the sequencing workflow:

Prepare for your experiment You will need to:

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

__Library preparation__ You will need to:
  • Repair the DNA and prepare the DNA ends for adapter attachment
  • Attach sequencing adapters supplied in the kit to the DNA ends
  • Prime the flow cell and load your DNA library into the flow cell

2020 10 09 LSK109 hamilton workflow v1 DS

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 to further analysis (this step is optional)
重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • Ligation Sequencing Kit XL (SQK-LSK109-XL)
  • FLO-PRO002 (R9.4.1) flow cells
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料
  • 1 µg （或 100-200 fmol） gDNA
  • 1.5-3 µg (or 150-300 fmol) high molecular weight genomic DNA for R10.3 flow cells
  • Ligation Sequencing Kit XL (SQK-LSK109-XL)
  • Flow Cell Priming Kit XL (EXP-FLP002-XL)
耗材
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • 供Oxford Nanopore Technologies®连接测序使用的NEBNext®配套模块（目录号E7180S或 E7180L），或使用以下三种NEBNext®产品：
  • NEBNext FFPE修复混合液（NEB，M6630）
  • NEBNext Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • NEBNext 快速连接模块（NEB，E6056）
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
  • Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
  • Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
  • Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)
  • Hamilton PCR ComfortLid (Cat# 814300)
  • Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
  • Roche Diagnotics MagNA Pure LC Medium Reagent Tubs 20 (Cat# 03004058001)
  • Sarstedt Inc Screw Cap Micro tube 2 ml, PP 1000/case (e.g. FisherScientific, Cat# NC0418367)
  • Thermo Scientific™ Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate (Thermo Scientific™, cat # AB0859)
仪器
  • 盛有冰的冰桶
  • 涡旋混匀仪
  • 微孔板离心机，如Fisherbrand™ 微孔板迷你离心机（Fisher Scientific, 11766427）
  • Hamilton NGS STAR 96 (NGS STAR with Multi-Probe Head 96)
  • Hamilton On-Deck Thermal Cycler (ODTC)
可选仪器
  • Agilent生物分析仪（或等效仪器）
  • Qubit fluorometer plate reader (or equivalent for QC check)

For this protocol, you will need 1 µg (or 100-200 fmol) gDNA.

Users can start with lower input quantities (down to 100 ng) if performing DNA fragmentation to increase the number of DNA molecules in the sample, or if amplifying the sample by PCR.

Input DNA

How to QC your input DNA

It is important to use a plate reader to ensure the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

Input file worklist

A worklist input Excel file is required prior to running the protocol on the Hamilton NGS STAR 96. These contain information regarding the appropriate number of samples and well identifiers.

Example:

Source_SampleID Source_Well Target_Well
Sample_01 A1 A1
Sample_02 B1 B2
Sample_03 C1 C1

Hamilton NGS STAR 96

This method has been tested and validated using the Hamilton NGS STAR 96 (with 8 channels and MPH96) including an on deck thermal cycler (ODTC). An option to not use the ODTC is available in the method. The protocol may require some fine tuning for the NGS STAR 96 setup and the temperature/humidity of the customer laboratory.

Please contact your Hamilton representative for further details.

Deck layout Ligation XL deck layout

  • ODTC: On-Deck Thermal Cycler Module
  • MIDI plates: Abgene™ 96 Well 0.8mL Polypropylene Deepwell Storage Plate
  • Troughs: Hamilton 20 ml Reagent Reservoirs
  • HSP plates: Bio-Rad Hard-Shell® 96-Well PCR Plates
  • Hamilton ComfortLid: Hamilton PCR ComfortLid

供Oxford Nanopore Technologies®连接测序使用的NEBNext®配套模块

对于新用户，我们建议购买供Oxford Nanopore Technologies®连接测序的 NEBNext® 配套模块 （目录号E7180S或E7180L）。该配套模块内包含所有与连接测序试剂盒配套使用的NEB试剂。

请注意：涉及扩增子测序的实验指南中，无需使用NEBNext FFPE修复混合液和NEBNext FFPE修复缓冲液。

Consumables and reagent quantities required:

Consumables X24 samples X48 samples X96 samples
Hamilton 50 µl CO-RE tips with filter 216 432 768
Hamilton 300 µl CO-RE tips with filter 451 694 1176
Hamilton 1000 µl CO-RE tips with filter 64 64 64
Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid 3 3 3
Hamilton PCR ComfortLid 1 1 1
Bio-Rad Hard-Shell® 96-Well PCR Plate 2 2 2
Roche Diagnostics MagNA Pure LC Medium Reagent Tubs 20 2 2 2
Sarstedt Inc Screw Cap Micro Tube 2ml 2 4 5
Abgene™ 96 Well 0.8mL Polypropylene Deepwell Storage Plate 2 2 2
Reagents/kits X24 samples X48 samples X96 samples
80% ethanol 16.5 ml 28 ml 51 ml
AMPure XP Beads 6.8 ml 9.7 ml 15.5 ml
Ligation Sequencing Kit XL (SQK-LSK109-XL) 1 kit 1 kit 2 kits
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S) 2 kits 3 kits 5 kits
Alternatively: - - -
NEBNext FFPE DNA Repair Mix (Cat# M6630L) 1 kit 1 kit 2 kits
NEBNext Ultra II End Repair/dA-Tailing Module (Cat# E7546L) 1 kit 2 kits 3 kits
NEBNext Quick Ligation Module (Cat# E6056L) 1 kit 2 kits 3 kits

Note: These are the number of kits required for one run through for the selected number of samples.

Ligation Sequencing Kit XL (SQK-LSK109-XL) contents

lsk109-xl v1

Name Acronym Cap colour No. of tubes Full volume (μl)
DNA CS DCS Yellow 1 52
Adapter Mix AMX Green 4 60
Ligation Buffer LNB Clear 4 300
L Fragment Buffer LFB White cap, orange stripe on label 4 6,000
S Fragment Buffer SFB Grey 4 6,000
Sequencing Buffer SQB Red 4 900
Elution Buffer EB Black 1 1,200
Loading Beads LB Pink 4 612

Flow Cell Priming Kit XL (EXP-FLP002-XL) contents

EXP-FLP002-XL

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Flush Buffer FB Blue 4 15,400
Flush Tether FLT Purple 4 400

3. 计算机要求及软件

PromethION 24/48 的IT配置要求

PromethION设备的硬件能够同时控制多达24个（适用于P24型号）或48个（适用于P48型号）测序实验，并采集数据。此外，设备借助高性能GPU技术，可以实时识别碱基。 请参阅 PromethION IT 配置要求文档，了解更多信息。

PromethION 2 Solo 的IT配置要求

作为一款小型台式测序设备，PromethION 2 Solo可独立或同时运行两张测序芯片。您只需将PromethION 2 Solo连接到GridION Mk1或符合最低技术规格要求的独立计算机，即可实现数据的实时采集和分析。欲了解更多信息，请参阅PromethION 2 Solo IT 配置要求文档。

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. DNA repair and end-prep

材料
  • 1.5-3 µg (or 150-300 fmol) high molecular weight genomic DNA for R10.3 flow cells
耗材
  • 无核酸酶水（如ThermoFisher，AM9937）
  • NEBNext FFPE DNA 修复混合液（NEB，M6630）
  • NEBNext® Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • 新制备的80%乙醇（用无核酸酶水配制）
  • Thermo Scientific™ Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate (Thermo Scientific™, cat # AB0859)
  • Sarstedt Inc Screw Cap Micro tube 2 ml, PP 1000/case (e.g. FisherScientific, Cat# NC0418367)
  • Roche Diagnotics MagNA Pure LC Medium Reagent Tubs 20 (Cat# 03004058001)
  • Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
  • Hamilton PCR ComfortLid (Cat# 814300)
  • Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)
  • Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
  • Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
  • Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
仪器
  • 盛有冰的冰桶
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P100 移液枪和枪头
  • P10 移液枪和枪头
  • 微孔板离心机，如Fisherbrand™ 微孔板迷你离心机（Fisher Scientific, 11766427）
  • 涡旋混匀仪

Consumables and equipment quantities:

Consumable/equipment X24 samples X48 samples X96 samples
Hamilton 50 µl CO-RE tips with filter 96 192 384
Hamilton 300 µl CO-RE tips with filter 201 298 490
Hamilton 1000 µl CO-RE tips with filter 32 32 32
Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid 2 (1 EtOH & H20) 2 (1 EtOH & H20) 2 (1 EtOH & H20)
Hamilton PCR ComfortLid 1 1 1
Bio-Rad Hard-Shell® 96-Well PCR Plate 1 (1 input sample & 1 end prepped sample) 1 (1 input sample & 1 end prepped sample) 1 (1 input sample & 1 end prepped sample)
Hamilton 20 ml Reagent Reservoirs 1 1 1
Sarstedt Inc Screw Cap Micro Tube 2 ml 1 2 2
Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate 1 1 1

Reagents quantities:

Reagents X24 samples X48 samples X96 samples
80% ethanol 16.5 ml 28 ml 51 ml
AMPure XP Beads 3.7 ml 5.4 ml 8.9 ml
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S)
regarding the reagents below		 2 tubes 3 tubes 5 tubes
Alternatively: - - -
NEBNext FFPE DNA Repair Buffer 1 tube 1 tube 1 tube
NEBNext FFPE DNA Repair Mix 1 tube 1 tube 2 tubes
Ultra II End-prep Reaction Buffer 1 tube 2 tubes 3 tubes
Ultra II End-prep Enzyme Mix 1 tube 1 tube 2 tubes

Note: Dead volumes are included.

重要

可选步骤：DNA片段化以及片段大小筛选

本实验手册不包含DNA片段化步骤。但在某些情况下，将样品片段化可能有助于您的实验。例如，当起始gDNA量较少时（100ng-500ng），将DNA片段化能扩充分子数量，从而提高通量。请参考：牛津纳米孔社区“Extraction methods”（提取方法）板块的 DNA片段化部分。

我们也提供了一些用于富集DNA样品中长片段的方法，请参考：牛津纳米孔社区“Extraction methods”(提取方法) 板块的 片段大小筛选部分。

根据生产厂家的说明准备NEBNext FFPE DNA 修复混合液和 NEBNext Ultra II 末端修复/ dA尾添加模块，并置于冰上。

为获得最优表现，NEB建议如下：

  1. 于冰上解冻所有试剂。

  2. 轻弹并/或翻转各管，确保各试剂充分混匀。
    注意： 请切勿涡旋振荡 FFPE DNA修复混合液或 Ultra II末端修复酶混合物。

  3. 同一日内首次打开一管试剂前，请务必先将该管试剂瞬时离心。

  4. Ultra II 末端修复缓冲液和 FFPE DNA 修复缓冲液内可能出现少量沉淀。待此两管液体回复至室温后，使用移液枪上下吹打数次，打散沉淀；然后涡旋振荡30秒，以确保沉淀充分溶解。
    注意： 请务必涡旋振荡混匀缓冲液。

  5. FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。

Prepare each DNA sample per well with nuclease-free water in the input plate.

  • Per sample, transfer 1 μg (or 100-200 fmol) of genomic DNA into a well of the input plate
  • Adjust the volume to 48 μl with nuclease-free water
  • Mix thoroughly by pipetting
  • Spin down briefly in a microfuge

Quantify 1 µl of each eluted sample using a Qubit fluorometer plate reader off deck.

Switch on the Hamilton NGS STAR 96 robot and open 'Hamilton Run Control' on the computer by clicking the icon:

Hamilton icon

Click 'File' and 'Open' to choose the method to run on the liquid handling robot.

Click 'Process01: DNA repair and end-prep' to start.

Capture1

Click 'Process02: DNA repair and end-prep clean-up' to stop the automated library preparation and quantify the samples before the adapter ligation step.

Capture2

重要

It is mandatory for users to have an MPH module installed and we recommend the use of an ODTC module.

Select whether an ODTC module is available to use in the run and select Yes to use the MPH (96 Head) module.

Capture3

Click 'Browse' to choose the Input File Worklist for the specific number of samples in the run and click 'OK'.

Capture4

An input file worklist for the number of samples in the run must be generated before the run. Example of an input file worklist:

Source_SampleID Source_Well Target_Well
Sample_01 A1 A1
Sample_02 B1 B1
Sample_03 C1 C1

Prepare the End Prep Mastermix with the following reagents according to the Hamilton user interface. Click either 'Yes' or 'No' to continue.

Note: It is user preference whether to print and save the instructions.

Reagent volumes for all sample numbers:

Reagent Volume X24 samples Volume X48 samples Volume X96 samples
NEBNext FFPE DNA Repair Buffer 106.6 µl 213.3 µl 414.8 µl
NEBNext FFPE DNA Repair Mix 60.9 µl 121.8 µl 237 µl
Ultra II End-prep Reaction Buffer 106.6 µl 213.3 µl 414.8 µl
Ultra II End-prep Enzyme Mix 91.4 µl 182.8 µl 355.6 µl
Total 365.6 µl 731.2 µl 1422.2 µl

Capture5

Insert the ComfortLid position as displayed on screen. Click 'Ok' to continue.

Capture6

Insert plates to their corresponding positions. Click 'Ok' to continue.

Capture7

Load a full deck of 50 µl tips into the positions on screen. Click 'Ok' to continue.

Capture8

Highlight the 50 µl tips available to use on the 'Edit Tip Count' window and click 'Ok' to continue.

Capture9

Load a full deck of 300 µl tips in the positions on screen. Click 'Ok' to continue.

Capture10

Highlight the 300 µl tips available to use on the 'Edit Tip Count' window and click 'Ok' to continue.

Capture11

Freshly prepare 80% ethanol in nuclease-free water in a trough.

Reagents Volume X24 samples Volume X48 samples Volume X96 samples
80% ethanol 16.5 ml 28 ml 51 ml

Insert the trough of 80% ethanol in the position on screen and click 'Ok' to continue.

Capture12

提示

If the consumables used for troughs are not barcoded, click 'Exclude' on all the selected troughs inserted in the robot and click 'Execute' to continue.

Capture13

Prepare the AMPure XP beads by vortexing and load the 20 ml trough with the volume required:

Reagents Volume X24 samples Volume X48 samples Volume X96 samples
Beads 3.7 ml 5.4 ml 8.9 ml
重要

Ensure the AMPure XP beads are well mixed before use by vortexing.

Insert the trough of AMPure XP beads and nuclease-free water in their positions on screen. Click 'Ok' to continue.

Capture14

提示

If the consumables used for troughs are not barcoded, click 'Exclude' on all the selected troughs inserted in the robot and click 'Execute' to continue.

Capture13

Load 1000 µl tips and insert the input plate of DNA samples into the position on screen. Click 'Ok' to continue.

Capture16

Highlight the 1000 µl tips available to use on the 'Edit Tip Count' window and click 'Ok' to continue.

Capture17

注意

Ensure the mastermix is well mixed and homogenous before loading the 2 ml Sarstedt tubes. Mixing in the robot is not effective.

Mix and insert the prepared End Prep Mastermix into the positions on screen.

Capture18

Click 'Ok' to start the DNA repair and end-prep automation process.

Once the automation process has finished, there will be an on screen prompt to unload the plate. Click 'Ok' to continue.

DNA repair and end prep

Quantify 1 µl of each eluted sample using a Qubit fluorometer plate reader off deck.

步骤结束

Take forward the repaired and end repaired DNA into the adapter ligation and clean-up step.

5. Adapter ligation and clean-up

材料
  • Adapter Mix (AMX)
  • 连接测序试剂盒内的连接缓冲液（LNB）
  • 长片段缓冲液（LFB）
  • Short Fragment Buffer (SFB)
  • Oxford Nanopore测序试剂盒中的洗脱缓冲液（EB）
耗材
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • NEBNext 快速连接模块（NEB，E6056）
  • Thermo Scientific™ Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate (Thermo Scientific™, cat # AB0859)
  • Sarstedt Inc Screw Cap Micro tube 2 ml, PP 1000/case (e.g. FisherScientific, Cat# NC0418367)
  • Roche Diagnotics MagNA Pure LC Medium Reagent Tubs 20 (Cat# 03004058001)
  • Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
  • Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)
  • Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
  • Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
  • Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
仪器
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P100 移液枪和枪头
  • P10 移液枪和枪头
  • Vortex mixer
  • 微孔板离心机，如Fisherbrand™ 微孔板迷你离心机（Fisher Scientific, 11766427）

Consumables and equipment quantities:

Consumable/equipment X24 samples X48 samples X96 samples
Hamilton 50 µl CO-RE tips with filter 120 240 384
Hamilton 300 µl CO-RE tips with filter 250 396 686
Hamilton 1000 µl CO-RE tips with filter 32 32 32
Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid 2 (1 L/SFB & 1 EB) 2 (1 L/SFB & 1 EB) 2 (1 L/SFB & 1 EB)
Bio-Rad Hard-Shell® 96-Well PCR Plate 1 1 1
Hamilton 20 ml Reagent Reservoirs 1 1 1
Sarstedt Inc Screw Cap Micro Tube 2 ml 1 2 3
Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate 1 1 1

Reagents quantities:

Reagents X24 samples X48 samples X96 samples
Adapter Mix (AMX) 2 tubes 4 tubes 8 tubes
Ligation Buffer (LNB) 2 tubes 4 tubes 8 tubes
Elution Buffer (EB) 1 bottle 1 bottle 1 bottle
Long Fragment Buffer (LFB) 2 bottles 4 bottles 8 bottles
Short Fragment Buffer (SFB) 2 bottles 4 bottles 8 bottles
AMPure XP Beads 3.1 ml 4.3 ml 6.6 ml
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S)
regarding the reagent below:		 2 tubes 3 tubes 5 tubes
Alternatively: - - -
Quick T4 DNA Ligase 1 tube 2 tubes 3 tubes

Note: Dead volumes are included.

重要

Although the recommended third-party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.

Spin down and store the Quick T4 Ligase on ice until use.

Spin down and combine all the required tubes of Adapter Mix (AMX) required, and place on ice.

Thaw the Ligation Buffer (LNB) at room temperature, spin down and combine all the required tubes. Place on ice immediately after thawing and mixing.

Thaw a bottle of Elution Buffer (EB) at room temperature, mix by vortexing and place on ice.

重要

接头连接后的纯化步骤，可通过选择不同缓冲液，按需富集大于3kb的DNA片段（LFB），或均等纯化所有大小的片段（SFB）。

  • 如若富集3kb或更长的DNA片段，请使用长片段缓冲液（LFB）
  • 如需保留所有大小的DNA片段，请使用短片段缓冲液（SFB）

To enrich for DNA fragments of 3 kb or longer, thaw the Long Fragment Buffer (LFB) at room temperature, mix by vortexing and combine all the required bottles before storing on ice.

To retain DNA fragments of all sizes, thaw the Short Fragment Buffer (SFB) at room temperature, mix by vortexing and combine all the required bottles before storing on ice.

Click 'Process03: Adapter ligation' to start.

Capture19

Click 'Process04: Adapter ligation and clean-up' to stop the automated library preparation and quantify the samples before sequencing.

Capture20

重要

It is mandatory for the MPH module to be installed on the liquid handling robot. Select 'Yes' to use the MPH (96 Head) module.

Capture21

Click 'Browse' to choose the Input File Worklist used during DNA repair and end-prep.

Capture4

Prepare the Adapter Ligation Mastermix with the following reagents according to the Hamilton user interface. Select either 'Yes' or 'No' to continue.

Note: It is user preference whether to print and save the instructions.

Reagent volumes for all sample numbers:

Reagent Volume X24 samples Volume X48 samples Volume X96 samples
Adapter Mix (AMX) 140.5 µl 281 µl 559.5 µl
Ligation Buffer (LNB) 702.5 µl 1405 µl 2797.5 µl
Quick T4 DNA Ligase 281 µl 562 µl 1119 µl

image (14)

注意

Ensure the mastermix is well mixed and homogenous before loading the 2 ml Sarstedt tubes. Mixing in the robot is not effective.

Insert plates to their corresponding positions on screen. Click 'Ok' to continue.

Capture24

Load a full deck of 50 µl tips into the positions on screen. Click 'Ok' to continue.

Capture25

Highlight the 50 µl tips available to use on the 'Edit Tip Count' window. Click 'Ok' to continue.

Capture26

Load a full deck of 300 µl tips in the positions on screen. Click 'Ok' to continue.

Capture27

Highlight the 300 µl tips available to use on the 'Edit Tip Count' window. Click 'Ok' to continue.

Capture28

Prepare the AMPure XP beads by vortexing and load the 20 ml trough with the volume required:

Reagents Volume X24 samples Volume X48 samples Volume X96 samples
Beads 3.1 ml 4.3 ml 6.6 ml
重要

Ensure the AMPure XP beads are well mixed before use by vortexing.

Insert troughs of AMPure XP beads, LFB/SFB and EB in the positions on screen. Click 'Ok' to continue.

Reagent Volume X24 samples Volume X48 samples Volume X96 samples
Long/Short Fragment Buffer 2 bottles 4 bottles 8 bottles
Elution Buffer 1 bottle 1 bottle 1 bottle

Capture29

提示

If the troughs are not barcoded, click 'Exclude' on all the selected troughs inserted in the robot and click 'Execute' to continue.

Capture13

Insert 1000 µl tips and the Clean End Prep Plate to the correct positions on screen. Click 'Ok' to continue.

Capture31

Highlight the 1000 µl tips available to use on the 'Edit Tip Count' window. Click 'Ok' to continue.

Capture32

Insert the prepared Adapter Ligation Mastermix into the positions on screen. Click 'Ok' to continue.

Capture33

注意

Ensure the mastermix is well mixed and homogenous before loading the 2 ml Sarstedt tubes. Mixing in the robot is not effective.

Once the automation process has finished, there will be an on screen prompt to unload the plate. Click 'Ok' to continue.

Adapter ligation and clean up2

Quantify 1 µl of each eluted sample using a Qubit fluorometer plate reader off deck.

步骤结束

Seal the plate once the library is prepared and store on ice until ready to load onto the flow cell.

We do not recommend running the liquid handling robot overnight as the plate must be sealed and stored on ice as soon as library preparation is finished.

重要

We recommend loading 5-50 fmol of final prepared library onto a flow cell.

Loading more than the recommended input can have a detrimental effect on output. Dilute the library in Elution Buffer (EB) if required. If you are using the Flongle for sample prep development, we recommend loading 3-20 fmol instead.

提示

Library storage recommendations

We recommend storing libraries at 4°C for short term storage or repeated use, for example, re-loading flow cells between washes. For single use and long term storage of more than 3 months, we recommend storing libraries at -80°C. For further information, please refer to the Library Stability Know-How document.

可选操作

If quantities allow, the libraries 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.

6. Priming and loading multiple flow cells on a PromethION

材料
  • Sequencing Buffer (SQB)
  • Loading Beads (LB)
  • Flow Cell Priming Kit XL (EXP-FLP002-XL)
耗材
  • PromethION 测序芯片
  • 1.5 ml Eppendorf DNA LoBind离心管
  • 2 ml Eppendorf DNA LoBind 离心管
仪器
  • PromethION 2 Solo 测序设备
  • PromethION测序设备
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P20 移液枪和枪头

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

重要

Scale up reagent volumes as needed.

Ensure to prepare enough reagents for the total number of flow cells being processed and to take into account extra volume required for pipetting errors.

提示

Each vial provides enough reagent for the preparation of 12 samples. Thaw the appropriate number of vials of each reagent.

Prepare the flow cell priming mix in a suitable vial for the number of flow cells to flush. Once combined, mix well by briefly vortexing.

Reagent Volume per flow cell
Flush Tether (FLT) 30 µl
Flush Buffer (FB) 1,170 µl
重要

将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。

对 PromethION 2 Solo，请按以下步骤为测序芯片上样：

  1. 将测序芯片平放在金属板上。

  2. 将测序芯片推入对接端口，直至金色引脚或绿色电路板不可见。

J2068 FC-into-P2-animation V5

对PromethION 24/48，将测序芯片插入相应卡槽的对接端口：

  1. 将测序芯片与连接器横竖对齐，以便顺利卡入。

  2. 用力下压芯片至卡槽，并确认卡夹位置归位。

Prom Flowcell Loading 1a 中文

Prom Flowcell Loading 1b 中文

重要

如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。

Screenshot 2021-04-08 at 12.08.37

If not already completed, perform a flow cell check on all flow cells.

Please refer to the Flow Cell Check protocol for further information.

顺时针滑动加液孔孔盖，将其打开。

Prom Flowcell Loading 2 中文

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。

在加液孔打开的状态下，按下述步骤吸取少量液体，同时避免引入气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  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。

Prom Flowcell Loading 3 中文

使用P1000移液枪向芯片的加液孔中加入500 µl芯片预处理溶液。加入过程中，请避免引入气泡。等待5分钟，与此同时，您可按以下步骤准备上样文库。

Prom Flowcell Loading 4 中文

Meanwhile, thoroughly mix the contents of the thawed Sequencing Buffer (SQB) and Loading Beads (LB) tube(s) by vortexing.

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 separate tube for each library, prepare for loading by adding the following reagents:

Reagent Volume
SQB 75 µl
LB 51 µl
DNA library 24 µl
Total 150 µl
可选操作

The Ligation Sequencing Kit XL kit is designed for users running multiple samples/flow cells. When handling multiple DNA preparations, the Sequencing Buffer (SQB) and Loading Beads (LB) can be combined in a master mix:

  1. Mix the Sequencing Buffer (SQB) and Loading Beads (LB) as described above, scaling up the final volume for the appropriate number of samples and adding up to 20% excess of each reagent.
  2. Mix the master mix by pipetting immediately before adding to the DNA samples.
  3. Pipette 126 µl of the master mix into each DNA sample-containing tube.
  4. Mix the sample by pipetting.

缓慢向芯片的加液口中加入500 µl预处理液，完成芯片的预处理。

Prom Flowcell Loading 5 中文

临上样前，用移液枪轻轻吹打混匀制备好的文库。

Using a P1000, insert the pipette tip into the inlet port and add 150 µl of library.

Step 6 v2

Close the valve to seal the inlet port and close the PromethION lid when ready.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

Step 7 V2

For multiple flow cell washing, use the same experiment name and identifying sample IDs for all runs to enable all flow cells to be paused simultaneously.

Screenshot 2023-02-14 114901

7. 数据采集和碱基识别

如何开始测序

在完成测序芯片的加样后，您即可在MinKNOW中启动测序实验。MinKNOW 软件负责仪器控制、数据采集以及实时碱基识别。有关设置和使用 MinKNOW 的详细信息，请参阅MinKNOW 实验指南。

您可以通过多种方式使用并设置MinKNOW：

  • 在直接或远程连接到测序设备的计算机上。
  • 直接在 GridION、MinION Mk1C 或 PromethION 24/48 测序设备上。

有关在测序设备上使用 MinKNOW 的更多信息，请参阅相应设备的用户手册：

在MinKNOW中启动测序：

1. 在 "开始 "（Start）页面上，选择 __开始测序__（Start Sequencing)。 start

2. 输入实验详情：例如实验名称，测序芯片位置及样本ID。 Grid start seq

3. 在"试剂盒"页面上，选择建库试剂盒。 kit selection

4. 配置测序实验参数，或保持“运行选项”和“分析”页面中的默认设置。

请注意： 如果在设置运行参数时关闭了碱基识别，您可在实验结束后，在MinKNOW中运行线下碱基识别。详情请参阅MinKNOW实验指南。

5. 在“输出”页面中，设置输出参数或保持默认设置。 step5c

6. 单击 "参数确认" 页面上的 开始 启动测序。 Step6

测序后数据分析

当于MinKNOW上完成测序后，您可按照“测序芯片的重复利用及回收”一节中的说明重复使用或返还测序芯片。

完成测序和碱基识别后，即可进行数据分析。有关碱基识别和后续分析选项的详细信息，请参阅数据分析文档。

在下游分析部分，我们将概述更多用于数据分析的选项。

8. 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。

9. 下游分析

下游分析

您可以选择以下几个途径来进一步分析经过碱基识别的数据：

1. EPI2ME 工作流程

Oxford Nanopore Technologies通过EPI2ME Labs平台提供了一系列针对高阶数据分析的生物信息学教程和工作流程。上述资源汇总于纳米孔社区的 EPI2ME Labs 板块。该平台通过描述性文字、生物信息学代码和示例数据，具象化地展示出我们的研究和应用团队发布在 GitHub 上的工作流程。

2. 科研分析工具

Oxford Nanopore Technologies的研发部门开发了许多分析工具，您可在Oxford Nanopore的 GitHub 资料库中找到。这些工具面向有一定经验的用户，并包含如何安装和运行软件的说明。工具以源代码形式提供，因此我们仅提供有限的技术支持。

3. 纳米孔社区用户开发的分析工具

如果以上工具仍无法为您提供解决研究问题的分析方法，请参考资源中心的生物信息学板块。该板块汇总了许多由纳米孔社区成员开发、且在Github上开源的、针对纳米孔数据的生信分析工具。请注意，Oxford Nanopore Technologies不为这些工具提供支持，也不能保证它们与测序所用的最新的化学试剂/软件配置兼容。

10. Issues during automation of library preparation

Please contact your automation vendor FAS and/or Nanopore FAS if you have any issues.

11. 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: 7/12/2023

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