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.

Cómo realizar un control de calidad del ADN de la muestra inicial

Es importante que la muestra de ADN cumpla con los requisitos de cantidad y calidad. Usar demasiado ADN, poco o de mala calidad (p. ej., que esté muy fragmentado, que contenga ARN o contaminantes químicos), puede afectar a la preparación de la biblioteca.

Para realizar un control de calidad en la muestra de ADN, consulte el protocolo Input DNA/ RNA QC

Contaminantes químicos

Dependiendo de cómo se extraiga el ADN de la muestra cruda, ciertos contaminantes químicos pueden permanecer en el ADN purificado, lo cual afecta la eficacia de la preparación de la biblioteca y la calidad de la secuenciación. Encontrará más información sobre contaminantes en la página Contaminants de la comunidad Nanopore.

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

The PromethION device contains all the hardware required to control up to 24 (for the P24 model) or 48 (for the P48 model) sequencing experiments and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the PromethION IT requirements document.

The PromethION 2 (P2) Solo is a device which directly connects into a GridION Mk1 or a stand-alone computer that meets the miminum specifications for real-time data streaming and analysis. Up to two PromethION flow cells can be can be run and each is independently addressable, meaning experiments can be run concurrently or individually. For information on the computer IT requirements, please see the PromethION 2 Solo IT requirements document.

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.

Antes de empezar el experimento de secuenciación, recomendamos verificar el número de poros disponibles, presentes en la celda de flujo. La comprobación deberá realizarse en las primeras 12 semanas desde su adquisición, si se trata de celdas de flujo MinION, GridION o PromethION, y en las primeras cuatro semanas tras la compra de celdas de flujo Flongle. Oxford Nanopore Technologies sustituirá cualquier celda de flujo con un número de poros inferior al indicado en la tabla siguiente, siempre y cuando el resultado se notifique dentro de los dos días siguientes a la comprobación y se hayan seguido las instrucciones de almacenamiento. Para verificar la celda de flujo, siga las instrucciones del documento Flow Cell Check.

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

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.

Para obtener una descripción completa del análisis de datos de nanoporos, que incluya distintas posibilidades para el análisis de identificación y postidentificicación de bases, consultar el documento Data Analysis.

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

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

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

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

Seguir las instrucciones del manual de usuario de GridION.

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

Seguir las instrucciones del manual de usuario de MinION Mk1C.

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

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

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

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

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.

También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.

Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.

También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.

Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.