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Multiplex Ligation Sequencing Kit V14 XL features
This kit is recommended for users who:
- Want to achieve median raw read accuracy of Q20+ (99%) and above.
- 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
- Want to achieve duplex basecalling. For more information, please see the Kit 14 sequencing and duplex basecalling info sheet.
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Introduction to the manual Multiplex Ligation Sequencing Kit V14 XL protocol
This protocol describes how to carry out native barcoding of genomic DNA using the Multiplex Ligation Sequencing Kit V14 XL (SQK-MLK114.96-XL). This kit is designed to enable low-plex sequencing.
This manual protocol outlines sample preparation with two options for low-plex sequencing:
- Two samples across one flow cell. Allowing users to sequence up to 96 samples across 48 flow cells, reducing cost per sample.
- Three samples across two flow cells. Allowing users to sequence up to 96 samples across 64 flow cells, maximising data output.
Using this protocol provides users with an easy workflow for whole genome sequencing (WGS), while enabling scalability options to best suit their sequencing requirements.
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
Prepare your library
You will need to:
- 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
- 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 software
- (Optional): Start the EPI2ME software and select a workflow for further analysis