Chapter 2 Overview of the workflow
2.1 Workflow steps
The laboratory workflow of the Earth Hologenome Initiative (EHI) is composed of six steps.
- Sample homogenisation and digestion: to break down the complex matrix of the sample and to disrupt cell walls.
- DNA extraction: to isolate DNA molecules from the rest of organic materials in the mixture and to remove chemical contaminants that may hinder further steps sensitive to inhibitors.
- DNA shearing: to achieve desired molecule sizes for optimal short-read sequencing.
- Sequencing library preparation: to convert fragmented DNA molecules into a format (library of DNA fragments) that is compatible with the sequencing platform.
- Sequencing library indexing: to produce copies (amplify) of the library using primers containing unique identifiers.
- Sequencing pool generation: to create a single sequencing pool containing multiple libraries in desired proportions.
2.2 General considerations
Automatisation
All the procedures implemented in the EHI laboratory have also been semi-automatised on a basic commercial pipetting and liquid handling robot (Opentrons OT-2). The procedure requires a workstation with a thermocycler and magnetic block.
Physical separation of laboratory environment
The steps 1-4 should be ran in a pre-PCR laboratory environment, where loads of environmental DNA are kept as low as possible to minimise sample contamination risk. As step 5 involves PCR amplification, any downstream procedure should be run in a post-PCR laboratory environment.