TITLE: A-Z of ancient DNA protocols for shotgun Illumina Next Generation Sequencing
AUTHORS: James Fellows Yates, Franziska Aron, Gunnar Neumann, Irina Velsko, Eirini Skourtanioti, Eleftheria Orfanou, Zandra Fagernäs, Raphaela Stahl, Aida Andrades Valtuena, Christina Warinner, Wolfgang Haak, Guido Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection contains established ancient DNA (aDNA) specific protocols for Illumina next generation sequencing. It covers sampling of skeletal remains for regions preserving higher amounts of aDNA, DNA extraction methods designed for retrieval of very short DNA fragments, and various library construction methods (including treatments for the 'removal' of typical aDNA damage). All protocols have been adapted after published protocols (see each for the corresponding original publication), and are used primarily in the Department of Archaeogenetics at the MPI-SHH (Jena, Germany).</div><div class = "text-block">All protocols in this collection can be used together, and in some cases swapped out with others. All protocols prior indexing are mostly performed in a dedicated ancient DNA laboratory - please see the guidelines section of each protocol for more information.</div><div class = "text-block">The order of protocols is as follows:</div><div class = "text-block"><span style = "font-weight:bold;">1. Sampling for aDNA</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>Sampling of </span><span style = "font-style:italic;">Pars petrosa (os temporale, '</span><span>'petrous bones') for human aDNA extraction</span></li><li style = "counter-reset:ol0;">Sampling of teeth for human and microbial (e.g. pathogen) aDNA extraction</li><li style = "counter-reset:ol0;">Sampling of dental calculus for oral microbiome aDNA extraction</li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">2. Retrieval of DNA</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span>Ancient DNA Extraction from Skeletal Material (based on the </span><a href="http://dx.doi.org/10.1073/pnas.1314445110" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Dabney et al. 2013 </span><span style = ":;font-style:italic;">Proc Natl Acad Sci U S A</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Ancient DNA extraction from dental calculus (based on the </span><a href="http://dx.doi.org/10.1073/pnas.1314445110" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Dabney et al. 2013 </span><span style = ":;font-style:italic;">Proc Natl Acad Sci U S A</span></a><span> with extra washing steps from </span><a href="https://dx.doi.org/10.1002/ajpa.22960" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ozga et al. 2016 </span><span style = ":;font-style:italic;">Am J Phys Anthropol</span></a><span style = ":;font-style:italic;">)</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">3. Library Preparation</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span>Library Adapter Preparation for Dual-Index Double Stranded DNA Illumina Sequencing (based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Non-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">requires 3.1</span><span>, based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Half-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">Variant of 3.2</span><span>, with partial damage removal treatment from </span><a href="https://dx.doi.org/10.1098/rstb.2013.0624" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Rohland et al. 2015 </span><span style = ":;font-style:italic;">Philos Trans R Soc Lond B Biol Sci</span></a><span style = ":;font-style:italic;">)</span></li><li style = "counter-reset:ol0;"><span>Full-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">Variant of 3.2</span><span>, with complete damage removal treatment from </span><a href="http://dx.doi.org/10.1093/nar/gkp1163" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Briggs et al. 2010 Nucleic Acids Res</span></a><span style = ":;">)</span></li><li style = "counter-reset:ol0;"><span>Dual-Indexing of aDNA libraries for Illumina Sequencing (based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4. Preparation for Sequencing </span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;">Amplification and Pooling for dual-indexed Illumina library for sequencing</li></ol></div><div class = "text-block"><span style = "font-weight:bold;">2. Retrieval of DNA</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>Ancient DNA Extraction from Skeletal Material (based on the </span><a href="http://dx.doi.org/10.1073/pnas.1314445110" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Dabney et al. 2013 </span><span style = ":;font-style:italic;">Proc Natl Acad Sci U S A</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Ancient DNA extraction from dental calculus (based on the </span><a href="http://dx.doi.org/10.1073/pnas.1314445110" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Dabney et al. 2013 </span><span style = ":;font-style:italic;">Proc Natl Acad Sci U S A</span></a><span> with extra washing steps from </span><a href="https://dx.doi.org/10.1002/ajpa.22960" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ozga et al. 2016 </span><span style = ":;font-style:italic;">Am J Phys Anthropol</span></a><span style = ":;font-style:italic;">)</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">3. Library Preparation</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span>Library Adapter Preparation for Dual-Index Double Stranded DNA Illumina Sequencing (based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Non-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">requires 3.1</span><span>, based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Half-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">Variant of 3.2</span><span>, with partial damage removal treatment from </span><a href="https://dx.doi.org/10.1098/rstb.2013.0624" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Rohland et al. 2015 </span><span style = ":;font-style:italic;">Philos Trans R Soc Lond B Biol Sci</span></a><span style = ":;font-style:italic;">)</span></li><li style = "counter-reset:ol0;"><span>Full-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">Variant of 3.2</span><span>, with complete damage removal treatment from </span><a href="http://dx.doi.org/10.1093/nar/gkp1163" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Briggs et al. 2010 Nucleic Acids Res</span></a><span style = ":;">)</span></li><li style = "counter-reset:ol0;"><span>Dual-Indexing of aDNA libraries for Illumina Sequencing (based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4. Preparation for Sequencing </span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;">Amplification and Pooling for dual-indexed Illumina library for sequencing</li></ol></div><div class = "text-block"><span style = "font-weight:bold;">3. Library Preparation</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>Library Adapter Preparation for Dual-Index Double Stranded DNA Illumina Sequencing (based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Non-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">requires 3.1</span><span>, based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"><span>Half-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">Variant of 3.2</span><span>, with partial damage removal treatment from </span><a href="https://dx.doi.org/10.1098/rstb.2013.0624" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Rohland et al. 2015 </span><span style = ":;font-style:italic;">Philos Trans R Soc Lond B Biol Sci</span></a><span style = ":;font-style:italic;">)</span></li><li style = "counter-reset:ol0;"><span>Full-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (</span><span style = "font-weight:bold;">Variant of 3.2</span><span>, with complete damage removal treatment from </span><a href="http://dx.doi.org/10.1093/nar/gkp1163" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Briggs et al. 2010 Nucleic Acids Res</span></a><span style = ":;">)</span></li><li style = "counter-reset:ol0;"><span>Dual-Indexing of aDNA libraries for Illumina Sequencing (based on </span><a href="http://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Meyer and Kircher 2010 </span><span style = ":;font-style:italic;">Cold Spring Harb. Protoc</span><span style = ":;font-style:italic;">.</span></a><span> protocol)</span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4. Preparation for Sequencing </span></li><li style = "counter-reset:ol0;"></li><li style = "counter-reset:ol0;">Amplification and Pooling for dual-indexed Illumina library for sequencing</li></ol></div><div class = "text-block"><span style = "font-weight:bold;">4. Preparation for Sequencing </span></div><div class = "text-block"><ol style = "list-style-type: decimal;"></ol></div></div>

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