It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated ...protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3' ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5' adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30-35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.
The use of hybridization capture has enabled a massive upscaling in sample sizes for ancient DNA studies, allowing the analysis of hundreds of skeletal remains or sediments in single studies. ...Nevertheless, demands in throughput continue to grow, and hybridization capture has become a limiting step in sample preparation due to the large consumption of reagents, consumables and time. Here, we explored the possibility of improving the economics of sample preparation via multiplex capture, that is, the hybridization capture of pools of double‐indexed ancient DNA libraries. We demonstrate that this strategy is feasible, at least for small genomic targets such as mitochondrial DNA, if the annealing temperature is increased and PCR cycles are limited in post‐capture amplification to avoid index swapping by jumping PCR, which manifests as cross‐contamination in resulting sequence data. We also show that the reamplification of double‐indexed libraries to PCR plateau before or after hybridization capture can sporadically lead to small, but detectable cross‐contamination even if libraries are amplified in separate reactions. We provide protocols for both manual capture and automated capture in 384‐well format that are compatible with single‐ and multiplex capture and effectively suppress cross‐contamination and artefact formation. Last, we provide a simple computational method for quantifying cross‐contamination due to index swapping in double‐indexed libraries, which we recommend using for routine quality checks in studies that are sensitive to cross‐contamination.
North Africa is a key region for understanding human history, but the genetic history of its people is largely unknown. We present genomic data from seven 15,000-year-old modern humans, attributed to ...the Iberomaurusian culture, from Morocco. We find a genetic affinity with early Holocene Near Easterners, best represented by Levantine Natufians, suggesting a pre-agricultural connection between Africa and the Near East. We do not find evidence for gene flow from Paleolithic Europeans to Late Pleistocene North Africans. The Taforalt individuals derive one-third of their ancestry from sub-Saharan Africans, best approximated by a mixture of genetic components preserved in present-day West and East Africans. Thus, we provide direct evidence for genetic interactions between modern humans across Africa and Eurasia in the Pleistocene.
Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to ...overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (
) the release of surface-bound DNA by phosphate buffer and (
) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.
Neandertal and Denisovan DNA from Pleistocene sediments Slon, Viviane; Hopfe, Charlotte; Weiß, Clemens L. ...
Science (American Association for the Advancement of Science),
05/2017, Letnik:
356, Številka:
6338
Journal Article, Web Resource
Recenzirano
Odprti dostop
Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using ...targeted enrichment of mitochondrial DNA, we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples, we detected Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave, we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility of detecting the presence of hominin groups at sites and in areas where no skeletal remains are found.
Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by ...technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals.
SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may ...introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches can be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics.
Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the ...molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30-75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.
The identification of bona fide microbial taxa in microbiomes derived from ancient and historical 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 for a set of samples that includes Neandertal remains, herbarium specimens and archaeological plant remains.
We show that sequencing DNA libraries enriched in molecules carrying uracils effectively amplifies age associated degradation patterns in microbial mixtures of ancient and historical origin. This facilitates the discovery of authentic ancient microbial taxa in cases where degradation patterns are difficult to detect due to large sequence divergence in microbial mixtures. Additionally, the relative enrichment of taxa in the uracil enriched fraction can help to identify bona fide ancient microbial taxa that could be missed using a more targeted approach.
Our experiments show, that in addition to its use in enriching authentic endogenous DNA of organisms of interest, the selective enrichment of damaged DNA molecules can be a valuable tool in the discovery of ancient microbial taxa.
The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly ...degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.
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