Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to the divergence of the modern O. phyllotis, supporting it as a distinct Pleistocene form of the Ototylomys genus. Hence, this rodent's tale suggests that the sister group to modern O. phyllotis arose during the Miocene-Pliocene, diversified during the Pleistocene and went extinct in the Holocene.
We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06- to 18.65-fold, to an apparent maximum threshold of ?80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.
Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic.
In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.
Quaternary plant ecology in much of the world has historically relied on morphological identification of macro- and microfossils from sediments of small freshwater lakes. Here, we report new protocols that reliably yield DNA sequence data from Holocene plant macrofossils and bulk lake sediment used to infer ecological change. This will allow changes in census populations, estimated from fossils and associated sediment, to be directly associated with population genetic changes.
The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.
Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGens WaxFree method and our in-house phenol-chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambions RecoverAll method recovered the most amplifiable RNA.
Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is undesirable. Two basic approaches to monitoring inhibition in such samples using real-time or quantitative PCR (qPCR) have been proposed. The first method analyzes the quantification cycle (Cq) deviation of a spiked internal positive control. The second method considers variations in reaction efficiency based on the slopes of individual amplification plots. In combining these methods, we observed increased Cq values together with reduced amplification efficiencies in some samples, as expected; however, deviations from this pattern in other samples support the use of both measurements. Repeat inhibition testing enables optimization of PCR facilitator combinations and sample dilution such that DNA yields and/or quantitative accuracy can be maximized in subsequent PCR runs. Although some trends were apparent within sample types, differences in inhibition levels, optimal reactions conditions, and expected recovery of DNA under these conditions suggest that all samples be routinely tested with this approach.
Gilbert et al. (Reports, 9 May 2008, p. 786) analyzed DNA from radiocarbon-dated paleofecal remains from Paisley Cave, Oregon, which ostensibly demonstrate a human presence in North America predating the well-established Clovis complex. We question the authenticity of their DNA results and argue that in the absence of intact stratigraphy and diagnostic artifacts, and in view of carbon isotope anomalies, the radiocarbon dates of the oldest specimens are unreliable.
Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples.
Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the Taq DNA polymerase during downstream applications. Here we present a simplified version of a protocol to extract DNA from paleofecal remains.
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