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In JoVE (1)
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Articles by Udo Stenzel in JoVE
प्राइमर एक्सटेंशन कैद: भारी अपमानित डीएनए स्रोत से लक्षित अनुक्रम रिट्रीवल
Adrian W. Briggs, Jeffrey M. Good, Richard E. Green, Johannes Krause, Tomislav Maricic, Udo Stenzel, Svante Pääbo
Max-Planck Institute for Evolutionary Anthropology, Leipzig
हम लक्षित प्राचीन डीएनए अनुक्रम पुनर्प्राप्ति, जो हम पांच Neandertal व्यक्तियों की पूरी mitochondrial जीनोम के पुनर्निर्माण के लिए इस्तेमाल किया की एक विधि प्रस्तुत करते हैं. वर्तमान दिन मनुष्यों के साथ इन दृश्यों की तुलना से पता चलता है कि Neandertals एक लंबी अवधि कम प्रभावी जनसंख्या के आकार का था.
Other articles by Udo Stenzel on PubMed
Nucleic Acids Research. 2007 | Pubmed ID: 17670798
High-throughput 454 DNA sequencing technology allows much faster and more cost-effective sequencing than traditional Sanger sequencing. However, the technology imposes inherent limitations on the number of samples that can be processed in parallel. Here we introduce parallel tagged sequencing (PTS), a simple, inexpensive and flexible barcoding technique that can be used for parallel sequencing any number and type of double-stranded nucleic acid samples. We demonstrate that PTS is particularly powerful for sequencing contiguous DNA fragments such as mtDNA genomes: in theory as many as 250 mammalian mtDNA genomes can be sequenced in a single GS FLX run. PTS dramatically increases the sequencing throughput of samples in parallel and thus fully mobilizes the resources of the 454 technology for targeted sequencing.
Proceedings of the National Academy of Sciences of the United States of America. Sep, 2007 | Pubmed ID: 17715061
High-throughput direct sequencing techniques have recently opened the possibility to sequence genomes from Pleistocene organisms. Here we analyze DNA sequences determined from a Neandertal, a mammoth, and a cave bear. We show that purines are overrepresented at positions adjacent to the breaks in the ancient DNA, suggesting that depurination has contributed to its degradation. We furthermore show that substitutions resulting from miscoding cytosine residues are vastly overrepresented in the DNA sequences and drastically clustered in the ends of the molecules, whereas other substitutions are rare. We present a model where the observed substitution patterns are used to estimate the rate of deamination of cytosine residues in single- and double-stranded portions of the DNA, the length of single-stranded ends, and the frequency of nicks. The results suggest that reliable genome sequences can be obtained from Pleistocene organisms.
Nature Protocols. 2008 | Pubmed ID: 18274529
Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.
Bioinformatics (Oxford, England). Jul, 2008 | Pubmed ID: 18467344
We present a tool suited for searching for many short nucleotide sequences in large databases, allowing for a predefined number of gaps and mismatches. The commandline-driven program implements a non-deterministic automata matching algorithm on a keyword tree of the search strings. Both queries with and without ambiguity codes can be searched. Search time is short for perfect matches, and retrieval time rises exponentially with the number of edits allowed. AVAILABILITY: The C++ source code for PatMaN is distributed under the GNU General Public License and has been tested on the GNU/Linux operating system. It is available from http://bioinf.eva.mpg.de/patman. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Cell. Aug, 2008 | Pubmed ID: 18692465
A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.
Science (New York, N.Y.). Jul, 2009 | Pubmed ID: 19608918
Analysis of Neandertal DNA holds great potential for investigating the population history of this group of hominins, but progress has been limited due to the rarity of samples and damaged state of the DNA. We present a method of targeted ancient DNA sequence retrieval that greatly reduces sample destruction and sequencing demands and use this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. We find that mtDNA genetic diversity in Neandertals that lived 38,000 to 70,000 years ago was approximately one-third of that in contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.
Direct Multiplex Sequencing (DMPS)--a Novel Method for Targeted High-throughput Sequencing of Ancient and Highly Degraded DNA
Genome Research. Oct, 2009 | Pubmed ID: 19635845
Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.
Genome Biology. 2009 | Pubmed ID: 19682367
The Illumina Genome Analyzer generates millions of short sequencing reads. We present Ibis (Improved base identification system), an accurate, fast and easy-to-use base caller that significantly reduces the error rate and increases the output of usable reads. Ibis is faster and more robust with respect to chemistry and technology than other publicly available packages. Ibis is freely available under the GPL from http://bioinf.eva.mpg.de/Ibis/.
Nucleic Acids Research. Apr, 2010 | Pubmed ID: 20028723
DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil-DNA-glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.
Genome Biology. 2010 | Pubmed ID: 20441577
High-throughput sequencing technologies have opened up a new avenue for studying extinct organisms. Here we identify and quantify biases introduced by particular characteristics of ancient DNA samples. These analyses demonstrate the importance of closely related genomic sequence for correctly identifying and classifying bona fide endogenous DNA fragments. We show that more accurate genome divergence estimates from ancient DNA sequence can be attained using at least two outgroup genomes and appropriate filtering.
Science (New York, N.Y.). May, 2010 | Pubmed ID: 20448178
Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the Neandertal genome to the genomes of five present-day humans from different parts of the world identify a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.
Road Blocks on Paleogenomes--polymerase Extension Profiling Reveals the Frequency of Blocking Lesions in Ancient DNA
Nucleic Acids Research. Sep, 2010 | Pubmed ID: 20587499
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.
Nature. Dec, 2010 | Pubmed ID: 21179161
Using DNA extracted from a finger bone found in Denisova Cave in southern Siberia, we have sequenced the genome of an archaic hominin to about 1.9-fold coverage. This individual is from a group that shares a common origin with Neanderthals. This population was not involved in the putative gene flow from Neanderthals into Eurasians; however, the data suggest that it contributed 4-6% of its genetic material to the genomes of present-day Melanesians. We designate this hominin population 'Denisovans' and suggest that it may have been widespread in Asia during the Late Pleistocene epoch. A tooth found in Denisova Cave carries a mitochondrial genome highly similar to that of the finger bone. This tooth shares no derived morphological features with Neanderthals or modern humans, further indicating that Denisovans have an evolutionary history distinct from Neanderthals and modern humans.