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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.
- Would like to utilise upstream processes such as size selection, whole genome amplification, or enrichment for long reads.
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Introduction to the whole genome amplification protocol
This protocol describes how to carry out whole genome amplification (WGA) of genomic DNA using the Ligation Sequencing Kit (SQK-LSK114) and the QIAGEN REPLI-g Midi kit.
Please note, the whole genome amplification step described in this protocol is based off the methods described in the REPLI-g® Mini/Midi Handbook. Please refer to the QIAGEN documentation for additional information.
This protocol uses the multiple displacement amplification (MDA) method with the QIAGEN kit to amplify as little as 50 pg of bacterial DNA to yield up to 40 ug DNA. T7 Endonuclease I treatment is performed to resolve the hyperbranched structure of the WGA product and to improve read quality (Qscore) and flow cell output. It is important to note, however, that by using this method some amplification bias can be introduced in the MDA reaction.
Please note, using this protocol will result in shorter fragment lengths and lower flow cell output than preparing a DNA library using the standard Ligation Sequencing DNA V14 protocol.
Please refer to our Sequencing products of multiple displacement amplification (MDA) know-how document for more information on the available methods.
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
Library preparation
You will need to:
- Amplify the genomic DNA using random hexamer primers
- Digest the amplified DNA with T7 Endonuclease I to remove branching, and size-select for longer fragments using AMPure XP beads
- 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
- Start the EPI2ME software and select a workflow for further analysis (this step is optional)
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Increasing flow cell output with PCR amplification
In instances where flow cell output is reduced when sequencing the products of MDA using the ligation-based protocol (<10 Gb from a MinION flow cell), we have found that performing a PCR amplification using the Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24) can recover flow cell output.
We recommend taking 5 ng of the MDA amplified sample from the whole genome amplification step described in this protocol and using it as input for the Rapid sequencing DNA - PCR Barcoding Kit 24 V14 (SQK-RPB114.24) protocol to perform additional PCR amplification.
Please note, amplification bias could potentially be exacerbated with additional PCR, and some GC-bias can be introduced, leading to a slight reduction in coverage at the extremes of GC-content.
For more information refer to the info sheet: Sequencing products of multiple displacement amplification (MDA).