-
Exome sequencing protocol features
Use this protocol if you:
- Are not interested in analysing the entire genome
- Want greater depth of coverage of a specific exonic region
- Want to analyse a large number of target regions
-
Introduction to exome sequencing
This protocol describes how to carry out exome sequencing using the Ligation Sequencing Kit (SQK-LSK109) and the Agilent SureSelect method.
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
- 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, or use minimap2 to align the reads to the human reference genome. -
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 g-TUBE
- 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
- LongAMP Taq is used for amplification of the DNA post-hybridisation