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Ligation Sequencing Kit V14 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.
- Require control over read length.
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Introduction to the protocol for sequencing 10 kb human DNA on PromethION using the Ligation Sequencing Kit V14 (SQK-LSK114)
This protocol describes an end-to-end process of extracting human genomic DNA using QIAGEN Puregene Cell Kit and DNA shearing using the Covaris g-TUBE™ to obtain an N50 of 10 kb read lengths. The Ligation Sequencing Kit V14 (SQK-LSK114) is used to prepare the DNA for sequencing. Data is basecalled by MinKNOW and the aligned BAM output data is analysed using the wf-human-variation workflow which uses Sniffles2, Clair3 and modkit software to call structural variants (SVs), single nucleotide polymorphisms (SNPs) and for reporting DNA methylation.
We recommend this protocol for users wanting an interaction-free sequencing run and high output from one DNA library, generating ~90 Gb of passed reads. An interaction-free sequencing run means no interaction by washing and re-loading of the flow cell with another DNA library is required during sequencing. Data generated can be used for analysing sequencing data for methylation calling and detecting single nucleotide polymorphisms (SNPs). This protocol has been optimised for output for the analysis of a genome at 30X coverage. However, SNP, SV and methylation detection can be performed with less coverage but 30X is optimal between sequencing performance whilst also achieving high reproducibility and robust detection.
For new users, we recommend completing a Lambda control experiment to become familiar with the Ligation Sequencing protocol with the Control Expansion (EXP-CTL001) prior to your first ligation experiment.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA using the QIAGEN Puregene Cell Kit, 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
Library preparationYou will need to:
- Shear your DNA to approximately 10 kb read lengths
- 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.
- Analyse data using the wf-human-variation workflow.
- Extract your DNA using the QIAGEN Puregene Cell Kit, and check its length, quantity and purity.