Single-stranded DNA library preparation from highly degraded DNA using<i>T4</i>DNA ligase

Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias

doi:10.1093/nar/gkx033

Cited by 210 publications

(246 citation statements)

References 39 publications

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“…In agreement with previous observations (Bennett et al 2014;Wales et al 2015;Gansauge et al 2017), the informative sequence content is substantially lower in the double-stranded than the single-stranded libraries (Supplemental Table S4; Supplemental Fig. S13).…”

Section: Resultssupporting

confidence: 91%

“…Library preparation, quantification, and amplification DNA libraries were prepared from 1-, 3-, 9-, and 27-µL aliquots of each DNA extract using a recently published single-stranded library preparation method (Gansauge et al 2017) automated on a liquid handling system (Bravo NGS workstation B, Agilent Technologies). Aliquots of DNA extract prepared with buffer exchange were incubated at 95°C for 1 min to inactivate carry-over of proteinase K from the lysis buffer.…”

Section: Dna Extractionmentioning

confidence: 99%

“…Single-stranded libraries were amplified for 35 cycles using AccuPrime Pfx DNA polymerase (Thermo Fisher Scientific) under reaction conditions described elsewhere (Dabney and Meyer 2012) except that indices were introduced into both adapters ) and index primer concentration was increased to 1 µM. Double-stranded libraries were amplified using both AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific) and AccuPrime Pfx DNA polymerase as described in Gansauge et al (2017). Amplified libraries were purified using the MinElute PCR purification kit (Qiagen).…”

Section: Dna Extractionmentioning

confidence: 99%

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

Glocke

Meyer

2017

Genome Res.

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The number of DNA fragments surviving in ancient bones and teeth is known to decrease with fragment length. Recent genetic analyses of Middle Pleistocene remains have shown that the recovery of extremely short fragments can prove critical for successful retrieval of sequence information from particularly degraded ancient biological material. Current sample preparation techniques, however, are not optimized to recover DNA sequences from fragments shorter than ∼35 base pairs (bp). Here, we show that much shorter DNA fragments are present in ancient skeletal remains but lost during DNA extraction. We present a refined silica-based DNA extraction method that not only enables efficient recovery of molecules as short as 25 bp but also doubles the yield of sequences from longer fragments due to improved recovery of molecules with singlestrand breaks. Furthermore, we present strategies for monitoring inefficiencies in library preparation that may result from co-extraction of inhibitory substances during DNA extraction. The combination of DNA extraction and library preparation techniques described here substantially increases the yield of DNA sequences from ancient remains and provides access to a yet unexploited source of highly degraded DNA fragments. Our work may thus open the door for genetic analyses on even older material.

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Section: Resultssupporting

confidence: 91%

Section: Dna Extractionmentioning

confidence: 99%

Section: Dna Extractionmentioning

confidence: 99%

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

Glocke

Meyer

2017

Genome Res.

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“…The bone powder of Vi-207 and Vi-208 was treated with 0.5% hypochlorite solution before DNA extraction (34) to remove microbial and modern human DNA contamination. Next-generation sequencing libraries were prepared from 10 μL of each extract, using two single-stranded library preparation schemes: Vi-207 and Vi-208 with a previously published method (35), with modifications from Korlevi c et al (34), and Vi-*28 with an automated library preparation scheme (36). Libraries were amplified and labeled with a pair of unique index sequences (37,38).…”

Section: Methodsmentioning

confidence: 99%

Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition

Devièse

Karavanić

Comeskey

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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107

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Previous dating of the Vi-207 and Vi-208 Neanderthal remains from Vindija Cave (Croatia) led to the suggestion that Neanderthals survived there as recently as 28,000–29,000 B.P. Subsequent dating yielded older dates, interpreted as ages of at least ∼32,500 B.P. We have redated these same specimens using an approach based on the extraction of the amino acid hydroxyproline, using preparative high-performance liquid chromatography (Prep-HPLC). This method is more efficient in eliminating modern contamination in the bone collagen. The revised dates are older than 40,000 B.P., suggesting the Vindija Neanderthals did not live more recently than others across Europe, and probably predate the arrival of anatomically modern humans in Eastern Europe. We applied zooarchaeology by mass spectrometry (ZooMS) to find additional hominin remains. We identified one bone that is Neanderthal, based on its mitochondrial DNA, and dated it directly to 46,200 ± 1,500 B.P. We also attempted to date six early Upper Paleolithic bone points from stratigraphic units G1, Fd/d+G1 and Fd/d, Fd. One bone artifact gave a date of 29,500 ± 400 B.P., while the remainder yielded no collagen. We additionally dated animal bone samples from units G1 and G1–G3. These dates suggest a co-occurrence of early Upper Paleolithic osseous artifacts, particularly split-based points, alongside the remains of Neanderthals is a result of postdepositional mixing, rather than an association between the two groups, although more work is required to show this definitively.

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“…The inability to taxonomically assign the vast majority of reads from samples with low endogenous DNA reflects the incompleteness of the reference database compared to the diversity of the microbiomes in those samples. Additionally, ssDNA library preparation methods generate shorter reads [21,22], which are more difficult to map to a reference genome and to assign taxonomically to a nucleotide database. This is reflected in the higher percentage of reads mapped and assigned from the dsDNA library compared with shorter reads derived from both the U-depleted and U-enriched fraction ( Figure S2A-B).…”

mentioning

confidence: 99%

Mining ancient microbiomes using selective enrichment of damaged DNA molecules

Weiß

Gansauge

Aximu‐Petri

et al. 2018

Preprint

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The identification of bona fide microbial taxa in microbiomes derived from historic samples is complicated by the unavoidable mixture between DNA from ante-and post-mortem microbial colonizers. One possibility to distinguish between these sources of microbial DNA is querying for the presence of age-associated degradation patterns typical of ancient DNA (aDNA). The presence of uracils, resulting from cytosine deamination, has been detected ubiquitously in aDNA retrieved from diverse sources, and used as an authentication criterion. Here, we employ a library preparation method that separates molecules that carry uracils from those that do not in a set of samples that includes Neandertal remains, herbaria specimens and archaeological plant remains. We show that this method facilitates the discovery of authentic ancient microbial taxa, as it amplifies degradation patterns that would otherwise be difficult to detect in sequences from diverse microbial mixtures. Main textDNA retrieved from historical or ancient samples is a complex mixture of molecules that contains not only endogenous host DNA, but also DNA from microorganisms that were present ante-mortem or that colonized the tissue post-mortem [1]. Therefore, all ancient DNA (aDNA) shotgun sequencing projects are metagenomic in nature. While earlier aDNA research has mostly focused on the evolution of animals and plants [2,3], a growing number of studies are now centering on the identification and characterization of ancient pathogens and microbiomes . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Single-stranded DNA library preparation from highly degraded DNA usingT4DNA ligase

Cited by 210 publications

References 39 publications

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

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

Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition

Mining ancient microbiomes using selective enrichment of damaged DNA molecules

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