Road blocks on paleogenomes - polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

Although the last few years have seen great progress in DNA sequence retrieval from fossil specimens, some of the characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions, i.e. chemical alterations that cannot be bypassed by DNA polymerases and thus prevent amplification and subsequent sequencing of affected molecules. Some studies have concluded that the vast majority of ancient DNA molecules carry blocking lesions, suggesting that the removal, repair or bypass of blocking lesions might dramatically increase both the time depth and geographical range of specimens available for ancient DNA analysis. However, previous studies used very indirect detection methods that did not provide conclusive estimates on the frequency of blocking lesions in endogenous ancient DNA. We developed a new method, polymerase extension profiling (PEP), that directly reveals occurrences of polymerase stalling on DNA templates. By sequencing thousands of single primer extension products using PEP methodology, we have for the first time directly identified blocking lesions in ancient DNA on a single molecule level. Although we found clear evidence for blocking lesions in three out of four ancient samples, no more than 40% of the molecules were affected in any of the samples, indicating that such modifications are far less frequent in ancient DNA than previously thought

Assessing the preservation of cytosine methylation in ancient DNA from five prehistoric Native American populations

textCytosine methylation of CpG dinucleotides is an important epigenetic mark that regulates gene expression in humans. While methylation patterns in extant populations have been widely studied, few studies have attempted to analyze methylation in ancient DNA. Indeed, it was only recently shown that methyl groups can be preserved in ancient DNA. However, it is unknown how often methylation patterns can be recovered from ancient samples with preserved nuclear DNA. If they are frequently preserved, it may ultimately be possible to infer patterns of gene activity at the population level in ancient times. In this study, I assessed the preservation of cytosine methylation in ancient DNA from the remains of 30 prehistoric Native Americans from California, Illinois, Kentucky, and Mexico. These samples were previously shown to contain endogenous mitochondrial and nuclear DNA. I analyzed the cytosine methylation states of CpG-rich retrotransposons, which are epigenetically inactivated by cytosine methylation in humans. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite. Bisulfite products were pyrosequenced, and C-to-T conversions at potentially methylated CpG dinucleotides were quantified. I found that cytosine methylation is readily recoverable from human remains with preserved nuclear DNA from various localities over the time depth tested (~6000 years). This study presents the first direct evidence of cytosine methylation in ancient human remains, and suggests that it may be possible to analyze patterns of gene activity in ancient populations.Anthropolog

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis

‘Dr Granville's mummy’ was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783–1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid–pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu

Genome-wide SNP typing of ancient DNA: Determination of hair and eye color of Bronze Age humans from their skeletal remains.

Objective A genome-wide high-throughput single nucleotide polymorphism (SNP) typing method was tested with respect of the applicability to ancient and degraded DNA. The results were compared to mini-sequencing data achieved through single base extension (SBE) typing. The SNPs chosen for the study allow to determine the hair colors and eye colors of humans. Material and methods The DNA samples were extracted from the skeletal remains of 59 human individuals dating back to the Late Bronze Age. The 3,000 years old bones had been discovered in the Lichtenstein Cave in Lower Saxony, Germany. The simultaneous typing of 24 SNPs for each of the ancient DNA samples was carried out using the 192.24 Dynamic Array (TM) by Fluidigm (R). Results Thirty-eight of the ancient samples (=64%) revealed full and reproducible SNP genotypes allowing hair and eye color phenotyping. In 10 samples (=17%) at least half of the SNPs were unambiguously determined, in 11 samples (=19%) the SNP typing failed. For 23 of the 59 individuals, a comparison of the SNP typing results with genotypes from an earlier performed SBE typing approach was possible. The comparison confirmed the full concordance of the results for 90% of the SNP typings. In the remaining 10% allelic dropouts were identified. Discussion The high genotyping success rate could be achieved by introducing modifications to the preamplification protocol mainly by increasing the DNA input and the amplification cycle number. The occurrence of allelic dropouts indicates that a further increase of DNA input to the preamplification step is desirable

Attempted DNA extraction from a Rancho La Brea Columbian mammoth (Mammuthus columbi): prospects for ancient DNA from asphalt deposits.

Fossil-bearing asphalt deposits are an understudied and potentially significant source of ancient DNA. Previous attempts to extract DNA from skeletons preserved at the Rancho La Brea tar pits in Los Angeles, California, have proven unsuccessful, but it is unclear whether this is due to a lack of endogenous DNA, or if the problem is caused by asphalt-mediated inhibition. In an attempt to test these hypotheses, a recently recovered Columbian mammoth (Mammuthus columbi) skeleton with an unusual pattern of asphalt impregnation was studied. Ultimately, none of the bone samples tested successfully amplified M. columbi DNA. Our work suggests that reagents typically used to remove asphalt from ancient samples also inhibit DNA extraction. Ultimately, we conclude that the probability of recovering ancient DNA from fossils in asphalt deposits is strongly (perhaps fatally) hindered by the organic compounds that permeate the bones and that at the Rancho La Brea tar pits, environmental conditions might not have been ideal for the general preservation of genetic material

Ancient DNA

DNA je genetski materijal svih organizama, to je molekula jednostavne građe, ali vrlo složene zadaće. Nosi uputu o svim funkcijama u našem tijelu, možemo reći nosi „znanja“ potrebna za život, jer u sebi sadrži informaciju potrebnu za funkcioniranje svih sustava u našem organizmu. Osim što nam omogućava život, molekula DNA nam prenosi gene naših predaka, zapise stare milijunima godina koji odolijevaju zubu vremena i čine se vječni. DNA nas povezuje s našom dubokom prošlosti i potiče našu svijest o samima sebi. Također zapis DNA, star milijunima godina, nam pruža odgovore na mnoge misterije iz prošlosti koji su ljude mučili dugo vremena. Postoji još mnogo toga što nam drevna DNA može ispričati, što tek trebamo otkriti i spoznati.DNA is the genetic material of all organisms. It is a molecule with a very simple structure, but very complex functions. It carries the instructions on all functions in our body, we can say that it carries knowledge needed for a life, because it contains information needed for a functioning of all systems in our organism. Beside it is needed for a life, DNA molecule carries genes of our ancestors, records millions of years old, which resist the ravages of time and seems internal. DNA connects us with our farther history and encourages our awareness of ourselves. As well, DNA code millions of years old, allovs us to solve the mysteries of the past, that people have struggled for a long time. There are still a lot to us of ancient DNA can tell, but we need to discover and recognize

Ancient fossil specimens of extinct species are genetically more distant to an outgroup than extant sister species are

There exists a remarkable correlation between genetic distance and time of species divergence as inferred from fossil records. This observation has provoked the molecular clock hypothesis. However, data inconsistent with the hypothesis have steadily accumulated in recent years from studies on extant organisms. Here the published DNA and protein sequences from ancient fossil specimens were examined to see if they would support the molecular clock hypothesis. The hypothesis predicts that ancient specimens cannot be genetically more distant to an outgroup than extant sister species are. Also, two distinct ancient specimens cannot be genetically more distant than their extant sister species are. The findings here did not support these predictions. Neanderthals are more distant to chimpanzees and gorillas than modern humans are. Dinosaurs are more distant to frogs than extant birds are. Mastodons are more distant to opossums than other placental mammals are. The genetic distance between dinosaurs and mastodons is greater than that between extant birds and mammals. Therefore, while the molecular clock hypothesis is consistent with some data from extant organisms, it has yet to find support from ancient fossils