-
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.
-
Introduction to the protocol for sequencing 30 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 the QIAGEN Puregene Cell Kit before performing size selection as well as shearing for a target N50 of 30 kb for input into the Ligation Sequencing Kit V14 (SQK-LSK114).
We have included instructions for washing and reloading the flow cell twice to maximise data output to reach ~90 Gb of passed reads using the Flow Cell Wash Kit (EXP-WSH004) and Sequencing Auxiliary Vials V14 (EXP-AUX003). 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 sequencing long N50s, such as 30 kb, for users comfortable monitoring their run to wash and re-load their flow cells when required to unblock pores and maintain high data acquisition. Data generated can be used for assembly and phasing applications, such as methylation and SNPs with phasing, and detecting SVs. This protocol has been optimised for output to generate ~90 Gb of passed reads for the analysis of a genome at 30X coverage. 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 size selection and sequencing kits, 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
Sample preparation
Using the outlined extraction method, extract the gDNA from your sample and perform size selection using the SFB Expansion (EXP-SFB001).
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 onto the flow cell
- Wash and reload your flow cell twice
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.