Benjamin Stielow - Fungi

Module IV: Taxon specific aspects of the barcoding pipeline

(Fungi)

29th NovemberPre-Conference Course

Benjamin Stielow

CBS Fungal Biodiversity Centre

Sample types

•Fresh specimens

•Herbarium specimens

•Actively growing culture

•Lyophilized culture

•Culture stored in liquid nitrogen

•Soil, other environmental samples…


Sample types

•Fresh specimens

•Herbarium specimens

•Actively growing culture (CBS barcoding)

•Lyophilized culture (CBS barcoding)

•Culture stored in liquid nitrogen (CBS barcoding)

•Soil, other environmental samples…


Sample types


DNA extraction

•DNA extraction from fungal samples is relatively simple

•Commercially available kits are well suitable• Genomed: Jetquick• Epicenter: Mastepure Yeast kit• Qiagen: DNeasy• EZNA: Forensic DNA kit• 96-well Acroprep plates


Challenges (PCR)

•Ribosomal genes are easy to amplify

•Protein coding genes can be difficult to amplify

•PCR setup can be time consuming (fine tuning)


Challenges (PCR)

•DNA elution volume can be critical (vs. PCR success)

•Use powerful polymerases (e.g. Takara HS)

•Use efficient PCR cyclers (e.g. Senso Quest)

•Use additives: BSA and DMSO

•Work in small sample volumes (12.5 μl)

•Dimension: 96 or 384 well format


DNA-lab and Sequencing unit

Project management

Sequence editing and validation

Private databases CBS

Strain selection

New Strains

DNA extraction

Collection

Stock database

PCR

Cycle sequencing

PCR check

Sequence facility

LIMS Raw data

Editing

Validation

Unpublished Barcode

Published Barcode

AutomatedBlast

Public Database(s)

Redo management

Release policy

DNA-Bank

Outlook


Thanks for your attention!

[email protected]


Education

Benjamin Stielow - Fungi