LABORATORY WORFLOW
1
Introduction
2
Overview of the workflow
2.1
Workflow steps
2.2
General considerations
Automatisation
Physical separation of laboratory environment
Instruments, plasticware and reagents
Instruments
Plasticware
3
Sample homogeneisation and digestion
3.1
Instruments, plasticware and reagents
Instruments
Plasticware
Reagents
3.2
Protocol
Faeces and swabs
Tissue samples
4
DNA extraction
4.1
Instruments, plasticware and reagents
Instruments
Plasticware
Stock reagents
4.2
Preparation of working reagents
Citrate buffer (0.1 M, ph 5.0)
Buffer B
Buffer C - DNA fraction
EBT buffer
Silica magnetic beads and buffer aliquots
4.3
Protocol
5
DNA shearing
5.1
Instruments, plasticware and reagents
Instruments
Plasticware
Stock reagents
5.2
Protocol
6
Library preparation
6.1
Instruments, plasticware and reagents
Instruments
Plasticware
Stock reagents
6.2
Preparation of working reagents
Reaction enhancer
6.3
Protocol
1. End-repair reaction
2. Ligation reaction
3. Fill-in reaction
4. Magnetic bead-based purification
7
Library indexing
7.1
Instruments, plasticware and reagents
Instruments
Plasticware
Stock reagents
7.2
Protocol
1. Library QC qPCR
2. PCR amplification
3. Magnetic bead-based purification
8
Library pooling
8.1
Instruments, plasticware and reagents
Instruments
Plasticware
Stock reagents
8.2
Protocol
1. Sample pooling
2. Final magnetic bead-based purification
References
Earth Hologenome Initiative
Bioinformatic workflow
Analysis workflow
The Earth Hologenome Initiative Laboratory Workflow
References