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Introduction to the low input protocol
This protocol describes how to carry out sequencing using a low starting input with the PCR Expansion (EXP-PCA001). The protocol outlines how ligate the PCR Adapter onto the gDNA fragment ends before PCR amplification. For amplicon DNA inputs, we recommend using a tailed primer approach and starting from the PCR step before preparing the sample with the Ligation Sequencing Kit V14 (SQK-LSK114).
It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
Note: For amplicon inputs, first-round PCR product with the following tailed primers are required. Please see the equipment and consumables page for further information.
5’ TTTCTGTTGGTGCTGATATTGC-[ project-specific forward primer sequence ] 3’
5’ ACTTGCCTGTCGCTCTATCTTC-[ project-specific reverse primer sequence ] 3’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
Experiment workflow
The table below is an overview of the steps required in the sample and library preparation, including timings and optional stopping points.
Library preparation Process Time Stop option For gDNA: DNA end-prep
or
For amplicons: Incorporate tailed primersPrepare the DNA ends for PCR adapter attachment
or
Perform a round of PCR to incoporate tailed primers35 minutes 4°C overnight PCR adapter ligation and amplification Amplify the DNA using the PCR primers 45 minutes 4°C overnight End-prep Repair the DNA and prepare the DNA ends for adapter attachment 35 minutes 4°C overnight Adapter ligation and clean-up Attach the sequencing adapters to the DNA ends 20 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell
-80°C for single-use, long-term storage.
We strongly recommend sequencing your library as soon as it is adapted.Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes Sequencing and analysis
You will need to:
- Start a sequencing run for SQK-LSK114 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)