METHODS IN PALAEOGENOMICS EVA CHOCHOLOVÁ LABORATORY OF BIOLOGICAL AND MOLECULAR ANTHROPOLOGY DEPARTMENT OF EXPERIMENTAL BIOLOGY QUIZ BREAKTHROUGH WITH HTS • Fragmentary aDNA – ideal for High Throughput Sequencing (Massive Parallel Sequencing, Next Generation Sequencing + Third Generation Sequencing) • ~ 30-60 bp impossible to target by PCR-based methods • Cost per base pair significantly lower in HTS compared to Sanger • Sanger still applicable but in different settings Lan and Lindqvist, 2018 DOI: 10.1007/13836_2017_7 Orlando et al., 2021 DOI: 10.1038/s43586-020-00011-0 Nishimura et al., 2022 DOI: 10.3390/v14061336 Nishimura et al., 2022 DOI: 10.3390/v14061336 PRESENT DNA CONTAMINATION CONTAMINATION HUMAN DNA Marciniak et al., 2015 DOI: 10.1016/j.jhevol.2014.11.003 Lan and Lindqvist, 2018 DOI: 10.1007/13836_2017_7 Lan and Lindqvist, 2018 DOI: 10.1007/13836_2017_7 HUMAN DNA - ENRICHMENT TARGET LOCUS TARGET LOCUS METABARCODING (16S rRNA…) METABARCODING (16S rRNA…) SHOTGUN Lan and Lindqvist, 2018 (DOI: 10.1007/13836_2017_7) Lan and Lindqvist, 2018 (DOI: 10.1007/13836_2017_7) Marciniak et al., 2015 DOI: 10.1016/j.jhevol.2014.11.003 Marciniak et al., 2015 DOI: 10.1016/j.jhevol.2014.11.003 Marciniak et al., 2015 DOI: 10.1016/j.jhevol.2014.11.003 Cappellini et al., 2018, DOI: 10.1146/annurev-biochem-062917-012002 Rohland et al., 2022, DOI: 10.1101/gr.276728.122 Marciniak et al., 2015 DOI: 10.1016/j.jhevol.2014.11.003 METABARCODING http://www.naturemetrics.co.uk METABARCODING http://www.sixthresearcher.com/amplicon-sequencing-and-high-throughput-genotyping-metagenomics/ METABARCODING http://www.sixthresearcher.com/amplicon-sequencing-and-high-throughput-genotyping-metagenomics/ http://www.sixthresearcher.com/amplicon-sequencing-and-high-throughput-genotyping-metagenomics/ Francioli et al., 2021, DOI: 10.3390/microorganisms9020361 Francioli et al., 2021, DOI: 10.3390/microorganisms9020361 • Cheaper – more samples, wider accessibility • Potentially better results for targeted analysis, lower yields, rare taxa… METABARCODING Warinner et al., 2014; DOI: 10.1038/ng.2906 METABARCODING Sawafuji et al., 2020; DOI: 10.1371/journal.pone.0226654 • Cheaper – more samples, wider accessibility • Potentially better results for targeted analysis, lower yields, rare taxa… METABARCODING Ziesemer et al., 2015; DOI: 10.1038/srep16498 • Cheaper – more samples, wider accessibility • Potentially better results for targeted analysis, lower yields, rare taxa… • PCR amplification bias (overrepresentation or underrepresentation of certain taxa) METABARCODING Ziesemer et al., 2015; DOI: 10.1038/srep16498 • Cheaper – more samples, wider accessibility • Potentially better results for targeted analysis, lower yields, rare taxa… • PCR amplification bias (overrepresentation or underrepresentation of certain taxa) Ziesemer et al., 2015; DOI: 10.1038/srep16498 Ziesemer et al., 2015; DOI: 10.1038/srep16498 CARBON FOOTPRINT COMBINING SOURCES Dussex et al., 2021; DOI: 10.1098/rspb.2021.1252 Limitations: • Sample size • Completeness of datasets • Temporal disconnection COMBINING SOURCES Dussex et al., 2021; DOI: 10.1098/rspb.2021.1252 UDG TREATMENT Hölz et al., 2019; DOI: 10.1038/s41598-019-54044-x Uracil DNA Glycosylase UDG TREATMENT aDNA sample Complete UDG treat. Complete UDG treat. on some extracts Partial UDG treat. Marciniak et al., 2015 DOI: 10.1016/j.jhevol.2014.11.003 EPIGENOMICS Orlando and Willerslev, 2014; DOI: 10.1126/science.1256515 Gokhman et al., 2017; DOI: 10.1093/molbev/msx211 THE CARP STORY https://dgg32.medium.com/carp-in-the-soil-1168818d2191 ADDITIONAL SOURCES ! • Only 5% endogeneous • Differences between library preparations etc • HTS • Metabarcoding • Enrichment why and how • Blocking primers in metabarcoding • Double indexing • Choosing markers for metabarcoding • Single stranded libraries better for aDNA • Carp