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Sequence capture protocol features
Use this protocol if you:
- Are not interested in analysing the entire genome
- Want greater depth of coverage of a specific region
- Want to analyse a large number of target regions
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Introduction to sequence capture
This protocol describes how to carry out sequence capture of genomic DNA using the Ligation Sequencing Kit (SQK-LSK110) and the Agilent SureSelect method for the kit used (e.g. SureSelect Human All Exon). Other kits of exome probes are available depending on the exome region coverage required.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- 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 runLibrary preparation
You will need to:
- Fragment your DNA (this step is optional)
- Ligate PCR adapters to the DNA ends and amplify the fragments
- Hybridise the DNA to probes provided in the Agilent SureSelect Exome kit, and perform a pulldown
- Elute and amplify the pulled-down fragments
- 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 cellSequencing 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 Human Exome mapping -
We recommend using the standard SureSelect Target Enrichment System protocol from Agilent Technologies with the following differences:
- The DNA in this protocol is sheared with dsDNA Fragmentase rather than in a Covaris microTUBE, and is incubated at 16° C instead of the 37° C recommended by NEB
- DNA purification in this protocol is carried out using Agencourt AMPure XP beads instead of QIAquick PCR purification
- DNA End-prep is carried out using the NEBNext Ultra II End Repair/dA-Tailing Module instead of the Agilent method
- Ligation of PCR adapters is carried out using the NEB Blunt/TA Ligase Master Mix instead of the Agilent method
- No desalting of the capture solution is necessary
- Long AMP Taq is used for amplification of the DNA post-hybridisation