Extending the spectrum of DNA sequences retrieved from ancient bones and teeth

Glocke, Isabelle; Meyer, Matthias

doi:10.1101/gr.219675.116

Cited by 90 publications

(91 citation statements)

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“…Other studies conducted with tooth and bone tissue also found no significant differences in average DNA fragment lengths recovered after modifying extraction procedures with extended digestion steps or bleach-based decontamination washes (Damgaard et al, 2015;Gamba et al, 2016;Korlevic et al, 2015). But a more recent report by Glocke and Meyer (2017) found that EDTA interferes with recovery of short DNA fragments. In general, reports comparing aDNA extraction methods have suggested that digestion time may be a strong influence on recovery rates and characteristics of endogenous aDNA.…”

Section: Discussionmentioning

confidence: 93%

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

Nieves-Colón

Ozga

Pestle

et al. 2017

Preprint

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Objectives:The tropics harbor a large part of the world's biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semitropical sites in Tanzania, Mexico, and Puerto Rico (N=12). Materials and Methods:All samples were extracted twice, built into double-stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step.Results: No significant difference was found in overall ancient DNA yields or post-mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality-filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments.Discussion: Both methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/184119 doi: bioRxiv preprint first posted online 2 Ancient DNA (aDNA) is a low quality and low quantity source of genetic material, which is highly susceptible to external contamination (Gilbert et al., 2006;Hofreiter et al., 2001;Pääbo, 1989). Due to the variety of taphonomic and diagenetic processes that take place after death, DNA decays exponentially once cell repair functions cease in biological tissues (Hofreiter et al., 2001). Consequently, most genetic information obtained from ancient samples is contained in small, degraded DNA fragments (Allentoft et al., 2012;Briggs et al., 2007;Dabney et al., 2013a). Because of this, until recently, aDNA studies have focused on short but informative fragments of the autosomal genome or, alternatively, on multicopy loci such as mitochondrial DNA (Ho & Gilbert, 2010). Recent advances in DNA extraction, target enrichment, and nextgeneration sequencing methods now allow the recovery of complete genomes from remains dating as far back in time as the early Holocene and Middle Pleistocene (Meyer et al., 2016;Meyer et al., 2014;Orlando et al., 2013).Despite these improvements in stretching the time depth for aDNA recovery...

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Section: Discussionmentioning

confidence: 93%

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

Nieves-Colón

Ozga

Pestle

et al. 2017

Preprint

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“…(Meyer et al, 2012) The single-strand approach has shown higher efficiency in the recovery of degraded and shorter DNA fragments. (Gansauge et al, 2017; Glocke & Meyer, 2017) Several recent studies have also shown that single-strand library preparation improved the recovery of cfDNA from cancer patients,(Snyder et al, 2016) transplantation recipients(Burnham et al, 2016) and maternal plasma. (Vong et al, 2017) It is of great significance to determine whether single-strand library preparation could indeed improve the performance of cfDNA analysis in clinical practice, especially in early-stage cancers.…”

Section: Introductionmentioning

confidence: 99%

Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

Liu

et al. 2018

Preprint

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30Analysis of cell-free DNA (cfDNA) is promising for broad applications in clinical 31 settings, but with significant bias towards late-stage cancers. Although recent studies have 32 discussed the diverse and degraded nature of cfDNA molecules, little is known about its 33 impact on the practice of cfDNA analysis. Here we reported a new targeted sequencing by 34 combining single-strand library preparation and target capture (SLHC-seq). By applying 35 the new technology in plasma cfDNA from pancreatic cancer patients, we achieved higher 36 efficiency in analysis of mutations than previously reported using other detection assays. 37 SLHC-seq rescued short or damaged cfDNA fragments along to increase the sensitivity 38 and accuracy of circulating-tumor DNA detection. Most importantly, we found that the 39 small mutant fragments are prevalent in early-stage patients, which provides strong 40 evidence for fragment size-based early detection of pancreatic cancer. Collectively, the 41 new pipeline enhanced our understanding of cfDNA biology and provide new insights for 42 liquid biopsy. 43 44 MAIN TEXT 45 65 sequencing.(Aravanis et al, 2017; Zill et al, 2015) Moreover, the noninvasive nature of cfDNA 66 analysis enables doctors to monitor disease progression.(Couraud et al, 2014; Murtaza et al, 2013; 67 Zill et al, 2015) However, cfDNA analysis using large-scale next-generation sequencing (NGS) 68 has been largely confined to advanced or metastatic cancers, in which the ctDNA ( circulating-69 tumor DNA ) fraction is higher and is more readily detectable.(Gorgannezhad et al, 2018; Haber 70 & Velculescu, 2014) Detection of ctDNA in early-stage cancers is still filled with cautionary tales 71 that highlight the technical challenges in this field. Recently, Velculescu et al reported improved 72 targeted error correction sequencing ( TEC-Seq ) as an efficient approach for the detection of 73 ctDNA in early-stage colorectal, ovarian, breast, and lung cancers, with a detection rate ranging 74 from 59% to 71%.(Phallen et al, 2017) The massive parallel sequencing approach provides an 75 applicable paradigm guiding cfDNA analysis in early-stage cancers. 76 However, the performance of existing approaches is limited in cfDNA analysis in early-77 stage pancreatic cancers. This limitation may be partially because of a lower ctDNA content in 78 the circulation of pancreatic cancers.(Aravanis et al, 2017) Moreover, the biological features of 79

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“…Sample pretreatment may also be unpredictable (Basler et al, 2017) and has even been contra-indicated for samples with very limited availability of bone material (Korlevi c et al, 2015). A third approach for increasing endogenous content is to utilize laboratory methods that target specific DNA fragment sizes, as reported for DNA extraction (Dabney et al, 2013;Glocke & Meyer, 2017) and some library preparation methods (Bennett et al, 2014). These increases presumably reflect substantial differences in the fragment length distributions of endogenous and contaminant DNA.…”

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confidence: 99%

Optimized DNA sampling of ancient bones using Computed Tomography scans

Alberti

González

Paijmans

et al. 2018

Molecular Ecology Resources

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The prevalence of contaminant microbial DNA in ancient bone samples represents the principal limiting factor for palaeogenomic studies, as it may comprise more than 99% of DNA molecules obtained. Efforts to exclude or reduce this contaminant fraction have been numerous but also variable in their success. Here, we present a simple but highly effective method to increase the relative proportion of endogenous molecules obtained from ancient bones. Using computed tomography (CT) scanning, we identify the densest region of a bone as optimal for sampling. This approach accurately identifies the densest internal regions of petrous bones, which are known to be a source of high-purity ancient DNA. For ancient long bones, CT scans reveal a high-density outermost layer, which has been routinely removed and discarded prior to DNA extraction. For almost all long bones investigated, we find that targeted sampling of this outermost layer provides an increase in endogenous DNA content over that obtained from softer, trabecular bone. This targeted sampling can produce as much as 50-fold increase in the proportion of endogenous DNA, providing a directly proportional reduction in sequencing costs for shotgun sequencing experiments. The observed increases in endogenous DNA proportion are not associated with any reduction in absolute endogenous molecule recovery. Although sampling the outermost layer can result in higher levels of human contamination, some bones were found to have more contamination associated with the internal bone structures. Our method is highly consistent, reproducible and applicable across a wide range of bone types, ages and species. We predict that this discovery will greatly extend the potential to study ancient populations and species in the genomics era.

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Extending the spectrum of DNA sequences retrieved from ancient bones and teeth

Cited by 90 publications

References 47 publications

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

Optimized DNA sampling of ancient bones using Computed Tomography scans

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