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Module IV: Taxon specific aspects of the barcoding pipeline (Fungi) 29 th November

CBS Fungal Biodiversity Centre. Module IV: Taxon specific aspects of the barcoding pipeline (Fungi) 29 th November Pre-Conference Course Benjamin Stielow. CBS Fungal Biodiversity Centre. Sample types Fresh specimens Herbarium specimens Actively growing culture Lyophilized culture

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Module IV: Taxon specific aspects of the barcoding pipeline (Fungi) 29 th November

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  1. CBS Fungal Biodiversity Centre Module IV: Taxon specific aspects of the barcoding pipeline (Fungi) 29th November Pre-Conference Course Benjamin Stielow

  2. CBS Fungal Biodiversity Centre • Sample types • Fresh specimens • Herbarium specimens • Actively growing culture • Lyophilized culture • Culture stored in liquid nitrogen • Soil, other environmental samples…

  3. CBS Fungal Biodiversity Centre • 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…

  4. CBS Fungal Biodiversity Centre Sample types

  5. CBS Fungal Biodiversity Centre • 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

  6. CBS Fungal Biodiversity Centre • Challenges (PCR) • Ribosomal genes are easy to amplify • Protein coding genes can be difficult to amplify • PCRsetup can be time consuming (fine tuning)

  7. CBS Fungal Biodiversity Centre • 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

  8. Project management Public Database(s) New Strains Strain selection Published Barcode Private databases CBS Collection Release policy DNA-Bank LIMS Stock database Unpublished Barcode Raw data Editing DNA extraction Cycle sequencing Automated Blast PCR PCR check Validation Sequence facility Redo management Sequenceediting and validation DNA-lab and Sequencing unit

  9. CBS Fungal Biodiversity Centre Outlook

  10. CBS Fungal Biodiversity Centre Thanks for your attention! b.stielow@cbs.knaw.nl

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