This kit is recommended for users who:

• Would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid handling robot
• Want to optimise their sequencing experiment for output
• Wish to low-plex samples for Whole Genome Sequencing (WGS)
• Need a PCR-free method of multiplexing to preserve additional information, such as base modifications
• Require control over read length
• Would like to utilise upstream processes, such as size selection or whole genome amplification

The Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL) is designed to enable low-plex sequencing. Oxford Nanopore Technologies has written an internal script which enables the liquid handling robot to carry out native barcoding of genomic DNA using this sequencing kit. We currently allow the multiplexing of two samples on a flow cell or three samples on two flow cells.

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 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

Automated library preparation

You will need to:

Off-deck:

• Prepare your sample input plate off-deck

On-deck:

• Repair the DNA, and prepare the DNA ends for adapter attachment
• Ligate Native barcodes supplied in the kit to the DNA ends
• Ligate sequencing adapters supplied in the kit to the DNA ends

Off-deck:

• Prime the flow cell, and load your DNA library into the flow cell

Sequencing

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
• Demultiplex barcoded reads in MinKNOW or the Guppy basecalling
• Start the EPI2ME software and select a workflow for further analysis (this step is optional)

96 samples with 2 samples per flow cell

Process	Minutes per step
End Repair and Adenylation	56
Native Barcode Ligation	75
Adapter Ligation	95
Total	226

96 samples with 3 samples across 2 flow cells

Process	Minutes per step
End Repair and Adenylation	56
Native Barcode Ligation	73
Adapter Ligation	78
Total	207

We recommend using 80 ng/µl per sample with a minimum of 15 µl per well when preparing the input plate.

DNA质控

选择符合质量和浓度要求的起始DNA至关重要的。使用过少或过多的DNA，或者质量较差的DNA（如，高度碎片化、含有RNA或化学污染物的DNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控实验指南 。

化学污染物

从原始样本中提取DNA的方法不同，可能会导致经纯化的DNA中所残留的化学污染物不同。这会影响文库的制备效率和测序质量。请在牛津纳米孔社区的 Contaminants（污染物）页面 了解更多信息。

Input workfiles are required prior to running this protocol on the Hamilton NGS STAR 96.

Oxford Nanopore Technologies will provide a template of the input workfiles in an Excel file.

• In the DATA tab of the file, there will be the template for the library preparation data
• In the INFO tab, there will be the template for the identification data

Library preparation data example:

Identification data example:

Output workfiles will be generated. Below are examples of the output workfiles after a run through of the method.

Library preparation data example:

Identification data example:

This method has been tested and validated using the Hamilton NGS STAR 96 (with Multi-Probe Head 96), Hamilton Ambion magnet adapter, Hamilton MTP landscape carrier and Hamilton On-Deck Thermal Cycler (ODTC).

Deck layout

The deck layout has been updated from the standard layout. Please see the "Prepare the deck" step of this protocol for more details.

For data tracking purposes, we have included the option to add user ID before starting any process. This can be filled using any method the user prefers.

We recommend using barcode stickers to track the input and output plates for data tracking. These can be tracked on the workfile and entered on the UI alongside the reagent lot barcodes when prompted.

This will contain the appropriate NEB reagents and the required volumes for the protocol on the Hamilton NGS STAR 96. For more information from NEB, please see "Find Products for Nanopore Sequencing".

Name	Acronym	Cap colour	Number of vials	Fill volume per vial (µl)
Adapter Mix II T	AMII T	Green	1	320
Sequencing Buffer II	SBII	Red	4	1,500
Loading Beads II	LBII	Pink	4	1,500
Loading Solution	LS	White cap, pink sticker	4	1,500
EDTA	EDTA	Clear	1	700
Elution Buffer	EB	15 ml bottle	1	10,000
Long Fragment Buffer	LFB	30 ml bottle	1	20,000
Flush Buffer XL	FB	30 ml bottle	6	15,500
Flush Tether	FLT	White cap, purple sticker	2	1,600
Native Barcodes	NB01-96	N/a	1 plate	8 µl per well

Consumables	No. of consumables for all conditions
Hamilton 50 µl CO-RE tips with filter	960
Hamilton 300 µl CO-RE tips with filter	960
Hamilton 1000 µ CO-RE tips with filter	96
Bio-Rad Hard-Shell® 96-well PCR Plate	8

This protocol requires the tip decks to be completely filled before starting a run. Partially filled tip decks will cause an error with the liquid handling robot.

We recommend using Hamilton tips for efficient liquid handling.

Note: Volumes for x48 samples will be available soon.

Full method

Reagents	x96 samples
80% ethanol	80 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
EDTA	1 vial
Native Barcode plate	1 plate
Adapter Mix F (AMII F)	1 vial
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl
Blunt/TA Ligase Master Mix	1210 µl
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

__Multiple steps combined:__ ### End Repair and Adenylation step to Native Barcode Ligation step

Reagents	x96 samples
80% ethanol	80 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate

### Native Barcode Ligation step to Adapter Ligation step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate
Adapter Mix F (AMII F)	1 vial
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

__Individual steps:__ #### End Repair step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl

### Native Barcode Ligation step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate

#### Adapter Ligation step

Reagents	x96 samples
AMPure XP Beads	15 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

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。

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。

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 this link.

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。

The deck layout has been updated from the standard NGS STAR 96 layout to improve the efficiency of the robot completing the protocol.

The changes will take approximately 3 minutes.

Final deck layout:

Consumables	No. of consumables for all conditions
Hamilton 50 µl CO-RE tips with filter	960
Hamilton 300 µl CO-RE tips with filter	960
Hamilton 1000 µ CO-RE tips with filter	96
Bio-Rad Hard-Shell® 96-well PCR Plate	8

Note: We recommend using Hamilton tips for efficient liquid handling.

Full method

Reagents	x96 samples
80% ethanol	80 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
EDTA	1 vial
Native Barcode plate	1 plate
Adapter Mix F (AMII F)	1 vial
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl
Blunt/TA Ligase Master Mix	1210 µl
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

Note: The following screenshots are an example of performing the full method for x96 samples.

Consumables	No. of consumables for all conditions
Hamilton 50 µl CO-RE tips with filter	960
Hamilton 300 µl CO-RE tips with filter	960
Hamilton 1000 µ CO-RE tips with filter	96
Bio-Rad Hard-Shell® 96-well PCR Plate	8

Note: We recommend using Hamilton tips for efficient liquid handling.

Note: Volumes for x48 samples will be available soon.

Multiple steps:

End Repair and Adenylation step to Native Barcode Ligation step

Reagents	x96 samples
80% ethanol	80 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate

### Native Barcode Ligation step to Adapter Ligation step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate
Adapter Mix F (AMII F)	1 vial
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

__Individual steps:__ ### End Repair step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl

### Native Barcode Ligation step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate

### Adapter Ligation step

Reagents	x96 samples
AMPure XP Beads	15 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

For an example of the dialogues illustrating how to load the deck, please see the "Complete automated library preparation" step.

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。

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 的说明操作：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 使用MinION Mk1B/Mk1D测序仪进行实时数据采集和碱基识别

请按照 MinION Mk1B 用户手册 或 MinION Mk1D 用户手册中的说明操作。

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 用户手册中的说明操作。

4. 使用GridION进行实时数据采集和碱基识别

请参照 GridION 用户手册 中的说明操作。

5. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 用户手册 或 PromethION 2 Solo 用户手册中的说明操作。

6. 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 中的说明操作：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。

There are several options for further analysing your basecalled data:

1. EPI2ME platform

The EPI2ME platform is a cloud-based data analysis service developed by Metrichor Ltd., a subsidiary of Oxford Nanopore Technologies. The EPI2ME platform offers a range of analysis workflows, e.g. for metagenomic identification, barcoding, alignment, and structural variant calling. The analysis requires no additional equipment or compute power, and provides an easy-to-interpret report with the results. For instructions on how to run an analysis workflow in EPI2ME, please follow the instructions in the EPI2ME protocol, beginning at the "Starting an EPI2ME workflow" step.

2. 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.

3. 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.

4. 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.

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。

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。