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

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

Fragment library length	Starting input
Very short (<1 kb)	200 fmol
Short (1-10 kb)	100–200 fmol
Long (>10 kb)	1 µg

For more information on sample input and flow cell loading amounts for our ligation sequencing protocols please visit our know-how document.

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

For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.

Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

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.

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.

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.

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within three months of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

Flow cell	Minimum number of active pores covered by warranty
Flongle Flow Cell	50
MinION/GridION Flow Cell	800
PromethION Flow Cell	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# 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.

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.

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).

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 on the Review page to start the sequencing run.

Kit 14 chemistry has improved duplex basecalling which requires rebasecalling on Dorado after simplex basecalling has been performed on MinKNOW.

For detailed information on setting up your sequencing run, for both simplex and duplex basecalling, please see the Kit 14 sequencing and duplex basecalling info sheet.

Note: When running Dorado, we recommend stopping other basecalling for the best performance by maximising memory available to Dorado. This can be stopped and restarted when Dorado has finished via the GUI on MinKNOW.

After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

In the Downstream analysis section, we outline further options for analysing your data.

There are several options for further analysing your basecalled data:

1. EPI2ME workflows

For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME, which are available in the EPI2ME section of the Community. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, that 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.

3. 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 Bioinformatics section of the Resource centre. 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.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.