This workshop is organised within the framework of the Belgian Network for DNA barcoding (BeBoL), which is financially supported by a FWO research network grant. It will focus on three aspects that are crucial for the analysis of DNA data from degraded biological material (e.g. old, ancient, museum or badly preserved specimens):
- DNA extraction methods for degraded material,
- library preparation and next-generation sequencing of fragmented DNA and
- quality control and interpretation of the results (through illustrated examples).
Date: 02-03 June 2016
Venue: Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium.
Registration: free but mandatory (only a limited number of participants will be able to join the demo in the wet lab on the 3rd of June). To register, please send an email to Gontran Sonet (gontran.sonet at naturalsciences.be) with your name, affiliation and your interest: talk only / demo in the wet lab only / both. Please register as soon as possible in order to allow us to book an appropriate auditorium.
Thursday 2 June 2016: lectures in the large auditorium (chair: Claudio Ottoni)
09:30 Registration and coffee
10:00 Claudio Ottoni (Department of Imaging and Pathology, University of Leuven, Leuven, Belgium)
• Overview: next generation sequencing of ancient DNA
• DNA extraction: ancient bones, teeth and soft tissues from mammals
• NGS technique: amplicon sequencing on an IonTorrent platform
• Example: animal domestication (pigs and cats)
11:00 Hernán Burbano (Research Group for Ancient Genomics and Evolution, Max Planck Institute for Developmental Biology, Tuebingen, Germany).
• Ancient DNA: biochemical characteristics and authenticity
• Temporal patterns of DNA and decay kinetics of DNA retrieved from herbarium specimens
• Extraction of ultra-short DNA molecules
• Colonization of new ecological niches by introduced species: Arabidopsis thaliana in North America
13:30 Katerina Guschanski (Evolutionary Biology Centre (EBC), Dept of Ecology and Genetics/Animal Ecology, Uppsala University, Uppsala, Sweden).
• DNA extraction: ancient DNA from teeth (Dabney et al. 2013).
• NGS technique: library preparation for NGS with enrichment of portions of the genomes (ex. mitochondrion) with a double-barcoding double-indexing strategy.
• Example: evolutionary processes that lead to biological diversity within and among populations and species (focus on nonhuman primates).
14:30 Danijela Popovic (Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland).
• DNA extraction: ancient DNA from bony scutes and museum samples of sturgeon
• NGS technique: amplicon sequencing on Illumina platforms and target enrichment of mitochondrial DNA.
• Example: phylogeography of European sturgeon.
16:00 Alfried P. Vogler (Faculty of Natural Sciences, Department of Life Sciences - Silwood Park, Imperial College, London, UK).
• DNA extraction: museum insects.
• NGS technique: metabarcoding of complex samples and whole mt genome sequencing.
• Example: beetle mtDNA phylogenomics.
17:00 End of the lectures
Friday 3 June 2016: practical demonstration in the wet lab
09:30 Katerina Guschanski & Tom van der Valk (Evolutionary Biology Centre (EBC), Dept of Ecology and Genetics/Animal Ecology, Uppsala University, Uppsala, Sweden).
N.B. Number of participants restricted to 20 (ca. 1 per research unit). Please note that participants will look at the experiments performed by the instructors. They will not handle any samples or reagents.
• DNA extraction of ancient DNA from teeth following the protocol of Dabney et al. (2013) and library preparation for NGS with a double-barcoding double-indexing strategy.
09:30 - 11:00
Extract samples that have been incubating during the night
11:00 - 12:00
Blunt-Ending ~30 minutes + 15 minutes incubation + minElute cleanup (~10 minutes)
12:00 - 12:30
Adapter ligation ~30 minutes
12:30 - 13:30
1 Hour Adapter ligation incubation* + LUNCH
*incubation should be minimal 30 minutes but can be longer, we should aim for not longer than 1 hour
13:30 - 13:45
minElute cleanup (~10 minutes)
13:45 - 14:15
Adapter fill-in ~30 minutes
14:15 - 15:00
40 minutes Adapter fill-in incubation + Collecting sample powder (cementum + dentin)
15:00 - 16:15
Indexing ~30 minutes + 30 minutes PCR + minElute cleanup (~10 minutes)
16:15 - 17:45
Bioanalyzer ~30 minutes + 1 hour run time*
In case we did not finish the drilling during the 40 minutes Adapter fill-in incubation we could finish it during the Bioanalyzer run.