-
Automated Multiplex Ligation Sequencing Kit XL features:
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
-
Introduction to the automated Multiplex Ligation Sequencing Kit XL protocol
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)
-
Timings
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