PromethION: Protocol

Ligation sequencing DNA V14 - automated Hamilton NGS STAR 96 (SQK-LSK114-XL) V GDA_9167_v114_revJ_24Aug2022

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 R10.4.1 flow cells

For Research Use Only

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 R10.4.1 flow cells

For Research Use Only

Document version: GDA_9167_v114_revJ_24Aug2022

1. Overview of the protocol

Ligation Sequencing Kit XL V14 features

This kit is recommended for users who:

Would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid-handling robot
Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above
Require control over read length
Would like to utilise upstream processes such as size selection, whole genome amplification, or enrichment 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。

重要

试剂盒14系列测序及互补双链碱基识别技术指南

"试剂盒14" 系列是 Oxford Nanopore Technologies 推出的最新产品系列，它提升了对互补双链DNA的碱基识别功能。与该系列配套的数据采集、碱基识别和分析工具与传统工具有所不同。更多信息，请参阅试剂盒14系列测序及互补双链碱基识别技术指南。我们强烈建议您在使用“试剂盒14”系列试剂测序及进行双链碱基识别前，阅读该技术指南。

Introduction to the automated Ligation Sequencing protocol for DNA

This protocol describes how to carry out sequencing of a DNA sample using the Ligation Sequencing Kit XL (SQK-LSK114-XL).

We have developed this automated protocol on the Hamilton NGS STAR 96 liquid handling robot. The majority of the process is automated with minimal hands-on time which is required for sample quantification and deck re-loading.

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

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

重要

Compatibility of this protocol

This protocol should only be used in combination with:

Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
R10.4.1 flow cells (FLO-PRO114M)
Flow Cell Wash Kit XL (EXP-WSH004-XL)

2. Equipment and consumables

材料

1 µg（或100-200 fmol）高分子量 DNA
如进行DNA片段化：100ng以上高分子量DNA
Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)

耗材

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）
Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
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)
Hamilton 20 ml Reagent Reservoirs (Cat# 96424-02)
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)

根据初始 DNA 样本长度调整样本起始量：

初始 DNA 样本长度	样本起始量
非常短 (<1 kb)	200 fmol
短 (1-10 kb)	100–200 fmol
长 (>10 kb)	1 µg

请参阅我们的专题技术文档，获取更多有关连接测序实验指南中样本起始量及测序芯片上样量的信息。

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

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。

第三方试剂

Oxford Nanopore Technologies推荐您使用本实验指南中提及的所有第三方试剂，并已对其加以验证。我们尚未对其它替代试剂进行测试。

我们建议您按制造商说明准备待用的第三方试剂.

重要

为确保高效接头（LA）连接，我们强烈建议您使用连接测序试剂盒V14中提供的连接缓冲液（LNB）而非其它第三方连接酶缓冲液。

重要

本试剂盒所用连接接头（LA）经过升级，不可与其它测序接头互换使用。

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
Hamilton 20 ml Reagent Reservoirs	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 V14 (SQK-LSK114-XL)	1 kit	1 kit	2 kits
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180L)	1 kits	1 kits	1 kits
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S)	-	1 kits	1 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 V14 (SQK-LSK114-XL) contents

Name	Acronym	Vial colour	Number of vials	Fill volume per vial (µl)
DNA Control Strand	DCS	Yellow	1	100
Ligation Adapter	LA	Green	1	320
Ligation Buffer	LNB	White	1	1,500
Elution Buffer	EB	White cap, black strip label	1	10,000
Long Fragment Buffer	LFB	White cap, orange strip label	2	20,000
Short Fragment Buffer	SFB	White cap, blue strip label	2	20,000
Library Beads	LIB	Pink	2	1,800
Library Solution	LIS	White cap, pink label	2	1,800
Sequencing Buffer	SB	Red	3	1,700
Flow Cell Flush	FCF	Clear	4	15,500
Flow Cell Tether	FCT	Purple	1	1,600

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

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 µg（或100-200 fmol）高分子量 DNA

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)
Hamilton 20 ml Reagent Reservoirs (Cat# 96424-02)
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# E7180L) regarding the reagents below	1 tubes	1 tubes	1 tubes
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S) regarding the reagents below	-	1 tubes	1 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

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。

为获得最优表现，NEB建议如下：

于冰上解冻所有试剂。
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。
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。
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。
FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。

Per sample, transfer 1 μg (or 100-200 fmol) of input 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

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.

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

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

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

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.

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.

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.

注意

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

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

连接接头（LA）
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

NEBNext 快速连接模块（NEB，E6056）
Agencourt AMPure XP beads (Beckman Coulter™, A63881)
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)
Hamilton 20 ml Reagent Reservoirs (Cat# 96424-02)
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）

重要

尽管第三方连接酶产品可能也附带缓冲液，但使用连接测序试剂盒中提供的连接缓冲液（LNB）时，连接接头（LA）的连接效率会更高。

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
Ligation adapter (LA)	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# E7180L) regarding the reagent below:	1 tubes	1 tubes	1 tubes
NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (Cat# E7180S) regarding the reagent below:	-	1 tubes	1 tubes
Alternatively:	-	-	-
Quick T4 DNA Ligase	1 tube	2 tubes	3 tubes

Note: Dead volumes are included.

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）

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.

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
Ligation Adapter (LA)	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

注意

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

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.

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

提示

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

注意

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

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.

文库片段长度	测序芯片上样量
非常短 (<1 kb)	100 fmol
短 (1-10 kb)	35–50 fmol
长 (>10 kb)	300 ng

请注意： 如您的文库产量低于我们的推荐值，请使用全部文库上样。

您可按需使用质量与摩尔数转换计算器，如 NEB 计算器。

重要

请根据下述建议向R10.4.1测序芯片中加入构建好的文库：

如希望获得 __高产出的单链数据__，请使用 35-50 fmol 的终文库上样，以确保孔利用率高于95%。请点击此处，查看如何计算孔利用率。

如希望获得 __双链数据__，请使用 10-20 fmol 的终文库上样。DNA上样量超过20 fmol会降低互补链序列的捕获率。

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

如DNA文库量足够，您可选择使用洗脱缓冲液（EB）稀释文库，再拆分上样至多张测序芯片。

根据测序芯片的数目，洗脱缓冲液的实际需求量可能会高于本试剂盒中该缓冲液的供应量。

6. PromethION 测序芯片的预处理及上样

材料

测序芯片冲洗液（FCF）
测序芯片系绳（FCT）
文库溶液（LIS）
文库颗粒（LIB）
测序缓冲液（SB）

耗材

PromethION 测序芯片
1.5 ml Eppendorf DNA LoBind 离心管

仪器

PromethION 2 Solo 测序设备
PromethION测序设备
PromethION 测序芯片遮光片
P1000 移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头

重要

本试剂盒仅兼容R10.4.1测序芯片（FLO-PRO114M）。

使用文库溶液

对大多数测序实验，我们建议您使用文库颗粒（LIB）给测序芯片上样。然而，对于粘稠的文库，借助文库颗粒上样可能会比较困难，此时使用文库溶液（LIS）可能更为合适。

试剂	体积（每张芯片）
测序芯片冲洗液 (FCF)	1170 µl
测序芯片系绳 (FCT)	30 µl
总体积	1200 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。

将测序芯片平放在金属板上。
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。

将测序芯片与连接器横竖对齐，以便顺利卡入。
用力下压芯片至卡槽，并确认卡夹位置归位。

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。

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P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
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。

重要

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

步骤结束

准备就绪后，合上PromethION设备上盖。

请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。

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)。

2. 输入实验详情：例如实验名称，测序芯片位置及样本ID。

3. 在"试剂盒"页面上，选择建库试剂盒。

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。

6. 单击 "参数确认" 页面上的开始启动测序。

互补双链碱基识别

Kit 14试剂改善了双链碱基识别方面的表现。如使用 Dorado 进行双链碱基识别，用户需首先在MinKNOW上进行单链碱基识别，然后再于Dorado上重新进行碱基识别，获得双链序列数据。

有关如何设置测序实验以进行单链和双链碱基识别的详细信息，请查阅 Kit 14 测序和互补双链碱基识别技术指南。

请注意： 当在Dorado上运行碱基识别时，我们建议您停止其他碱基识别操作，以最大化Dorado的可用内存，获得最佳性能。一旦Dorado在MinKNOW GUI上的运行结束，您可以重新启动其他操作。

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）

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。

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。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) 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.

Device:

London Calling 2025

所有产品

Documentation

Nanopore Learning

Ligation sequencing DNA V14 - automated Hamilton NGS STAR 96 (SQK-LSK114-XL)

Introduction to the protocol

Automated library preparation

测序及数据分析

Troubleshooting

Document versions

PromethION: Protocol

Ligation sequencing DNA V14 - automated Hamilton NGS STAR 96 (SQK-LSK114-XL) V GDA_9167_v114_revJ_24Aug2022

Contents

Introduction to the protocol

Automated library preparation

测序及数据分析

Troubleshooting

概览

1. Overview of the protocol

Ligation Sequencing Kit XL V14 features

重要

可选步骤：DNA片段化以及片段大小筛选

重要

试剂盒14系列测序及互补双链碱基识别技术指南

Introduction to the automated Ligation Sequencing protocol for DNA

重要

Compatibility of this protocol

2. Equipment and consumables

材料

耗材

仪器

可选仪器

根据初始 DNA 样本长度调整样本起始量：

Input DNA

How to QC your input DNA

Input file worklist

Hamilton NGS STAR 96

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

第三方试剂

重要

为确保高效接头（LA）连接，我们强烈建议您使用连接测序试剂盒V14中提供的连接缓冲液（LNB）而非其它第三方连接酶缓冲液。

重要

本试剂盒所用连接接头（LA）经过升级，不可与其它测序接头互换使用。

Consumables and reagent quantities required:

Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) contents

3. 计算机要求及软件

PromethION 24/48 的IT配置要求

PromethION 2 Solo 的IT配置要求

Software for nanopore sequencing

MinKNOW

EPI2ME (optional)

测序芯片质检

4. DNA repair and end-prep

材料

耗材

仪器

Consumables and equipment quantities:

Reagents quantities:

重要

可选步骤：DNA片段化以及片段大小筛选

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。

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

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:

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

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

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

重要

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

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

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

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

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

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

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

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

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

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

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

提示