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

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

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)

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.

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.

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

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

A los clientes que no conozcan la secuenciación por nanoporos, se les recomienda que adquieran el módulo de acompañamiento NEBNext® Ligation Sequencing para Oxford Nanopore Technologies® (NEB E7180S o E7180L), que contiene todos los reactivos NEB necesarios para usar con el kit Ligation Sequencing Kit.

Nótese que en los protocolos con amplicones no es necesario usar ni la mezcla de reparación de ADN NEBNext FFPE DNA Repair Mix, ni el tampón de reparación de ADN NEBNext FFPE DNA Repair Buffer.

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.

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

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

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 the EPI2ME Platform protocol.

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

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.

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.

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

MinKNOW can be used and set up to sequence in multiple ways:

• On a computer either direcly or remotely connected to a sequencing device.
• Directly on a GridION, MinION Mk1C or PromethION 24/48 sequencing device.

For more information on using MinKNOW on a sequencing device, please see the device user manuals:

• PromethION 24/48 user manual
• PromethION 2 Solo user manual

---

To start a sequencing run on MinKNOW:

1. Navigate to the start page and click Start sequencing.

2. Fill in your experiment details, such as name and flow cell position and sample ID.

3. Select the sequencing kit used in the library preparation on the Kit page.

4. Configure the sequencing and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.

Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.

5. Click Start to initiate the sequencing run.

Una vez la secuenciación ha finalizado, es posible reutilizar o devolver la celda de flujo, como se describe en la sección sobre Reutilización o retorno de celdas de flujo.

Tras secuenciar e identificar las bases, es posible analizar los datos. Si desea más información sobre las opciones de identificación de bases y de análisis posterior, consulte el documento Data Analysis.

En la sección Análisis posterior, se describen otras opciones para analizar los datos.

Existen varias opciones para completar el análisis de los datos de bases identificadas:

1. Flujos de trabajo de EPI2ME

A fin de realizar un análisis en profundidad de los datos, Oxford Nanopore Technologies ofrece una serie de tutoriales sobre bioinformática y flujos de trabajo, que están disponibles en EPI2ME. La plataforma proporciona un espacio donde los flujos de trabajo que depositan en GitHub nuestros equipos de Investigación y Aplicaciones, se pueden exponer con textos descriptivos, código bioinformático funcional y datos de ejemplo.

2. Herramientas de análisis para la investigación

Para realizar un análisis de datos más exhaustivo, Oxford Nanopore Technologies ofrece una serie de tutoriales y flujos de trabajo bioinformáticos, disponibles en EPI2ME Labs, que encontrará en la sección del mismo nombre de la comunidad Nanopore. La plataforma proporciona un espacio donde los proyectos que depositan en GitHub nuestros equipos de Investigación y Aplicaciones, se pueden exponer con textos descriptivos, código bioinformático funcional y datos de ejemplo.

3. Herramientas de análisis desarrolladas por la comunidad

Si no se proporciona en ninguno de los recursos anteriores un método de análisis que responda a las necesidades de investigación requeridas, consulte el centro de recursos Resource Centre y busque herramientas bioinformáticas para su aplicación. Varios miembros de la comunidad Nanopore han desarrollado sus propias herramientas y cartera de productos en desarrollo para analizar los datos de la secuenciación por nanoporos. La mayoría de ellas está disponible en GitHub. Oxford Nanopore Technologies no desarrolla ni mantiene esas herramientas y no garantiza que sean compatibles con la última configuración de química/programas informáticos.

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.