Chapter 2 Overview of the workflow

2.1 Workflow steps

The laboratory workflow of the Earth Hologenome Initiative (EHI) is composed of six steps.

  1. Sample homogenisation and digestion: to break down the complex matrix of the sample.
  2. DNA extraction: to isolate DNA molecules from the rest of organic materials in the mixture.
  3. DNA shearing: to achieve desired molecule sizes for optimal short-read sequencing.
  4. Sequencing library preparation: to convert fragmented DNA molecules into a format that is compatible with the sequencing platform.
  5. Sequencing library indexing: to amplify the library using primers containing unique identifiers.
  6. 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 workflow are automatisable in liquid handlers.

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.

Instruments, plasticware and reagents

Instruments

  • Laboratory centrifuge and microcentrifuge
  • Vortex
  • Single channel pipettes: 0.1-1 mL, 20-200 μL, 2-20 μL and 1-10 μL
  • Multi Channel pipettes: 20-200 μL and 1-10 μL

Plasticware

  • Filter-tips: 0.1-1 mL, 20-200 μL, 2-20 μL and 1-10 μL
  • Microcentrifuge Tubes: 1.5 mL, 2.0 mL and 5 mL
  • Centrifuge Tubes: 15 mL and 50 mL
  • Reagent Reservoir 50 mL