1) The authors determined the DNA sequence of the second hypervariable region (HVRII) of mitochondrial DNA from the Neandertal type specimen.
2) When combined with the previously published HVR1 sequence, phylogenetic analysis found the Neandertal mtDNA to fall outside the variation of contemporary human mtDNA sequences.
3) The estimated date of divergence between Neandertal and modern human mtDNA sequences was 465,000 years before present, with a confidence interval of 317,000 to 741,000 years. This supports the Neandertals having a separate evolutionary history from modern humans.
Ancient dna. would the real neandertal please stand up (cooper et al.)Kristian Pedersen
This document summarizes a study that analyzed mitochondrial DNA sequences recovered from Neandertal specimens and early modern human fossils. The study found:
1) Neandertal mtDNA sequences were not present in early modern humans or contemporary humans, suggesting a limited genetic contribution from Neandertals to modern humans.
2) However, the results depend on assumptions like the fossils actually containing ancient DNA, and more data is needed to definitively resolve the genetic relationship between Neandertals and modern humans.
3) Future studies using more specimens and nuclear DNA may provide more powerful tests of the extent of genetic exchange between Neandertals and early modern humans.
This document summarizes the history and development of CRISPR-Cas9 genome editing technology. It discusses how early approaches to targeted DNA cleavage relied on site-specific DNA recognition by oligonucleotides or proteins. Zinc finger nucleases and TAL effector nucleases were developed that allowed for genome editing but were difficult to design and validate. The discovery of CRISPR immune systems in bacteria provided the basis for a new approach using Cas9 guided by CRISPR RNA. Experiments showed Cas9 could be programmed to cleave DNA sequences using a simple RNA guide. This established the CRISPR-Cas9 system as an easy and efficient tool for genome editing that has enabled many new applications across biology.
Paired genes in stem cells shed new light on gene organization and regulation and epigenetic control of cardiogenesis. A study found that about 65% of long non-coding RNAs (lncRNAs) are transcribed in pairs with messenger RNAs (mRNAs) at active gene promoters in embryonic stem cells. Additionally, an experiment deactivated a lncRNA gene causing disruption in heart development and defects in mouse embryos, demonstrating that lncRNAs regulate tissue development and organogenesis. The discoveries provide new insights into gene transcription and the epigenetic roles of lncRNAs in development.
High-throughput RNA sequencing with Thermostable Group II Intron Reverse Tran...Douglas Wu
TGIRT is a thermostable group II intron reverse transcriptase that enables high-throughput RNA sequencing with several advantages over other reverse transcriptases. It recapitulates the relative abundance of transcripts, has higher strand-specificity, and provides more uniform gene coverage from 5' to 3' compared to TruSeq v3. TGIRT also enables simultaneous profiling of mRNA, lncRNA, and other structured small RNAs. Its applications include structured RNA-seq, plasma and exosomal RNA-seq for cancer diagnostics, and generation of long cDNAs.
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
Use of Thermostable Group II Intron Reverse Transcriptases (TGIRTs) for Singl...Douglas Wu
(1) Thermostable group II intron reverse transcriptase (TGIRT) enzymes enable efficient single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA (cfDNA) fragments in human plasma and other bodily fluids. (2) ssDNA-seq using TGIRT can identify epigenetic features in cfDNA fragments that provide information about the tissue of origin, with potential diagnostic applications. (3) TGIRT-based ssDNA-seq allows effective library construction from small amounts of degraded cfDNA samples.
Use of Thermostable Group II Intron Reverse Transcriptases (TGIRTs) for Singl...Douglas Wu
Thermostable group II intron reverse transcriptases (TGIRTs) enable efficient single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA fragments in human plasma in 2 hours with fewer reagents. TGIRT-seq analysis of nucleosome positioning and transcription factor occupancy in plasma DNA matches previous studies and can determine tissue-of-origin. While TGIRT-seq has a higher mismatch rate than other methods, it produces comparable genomic coverage and can detect nucleosome positioning, transcription factor binding sites, and tissue-of-origin from plasma DNA.
Ancient dna. would the real neandertal please stand up (cooper et al.)Kristian Pedersen
This document summarizes a study that analyzed mitochondrial DNA sequences recovered from Neandertal specimens and early modern human fossils. The study found:
1) Neandertal mtDNA sequences were not present in early modern humans or contemporary humans, suggesting a limited genetic contribution from Neandertals to modern humans.
2) However, the results depend on assumptions like the fossils actually containing ancient DNA, and more data is needed to definitively resolve the genetic relationship between Neandertals and modern humans.
3) Future studies using more specimens and nuclear DNA may provide more powerful tests of the extent of genetic exchange between Neandertals and early modern humans.
This document summarizes the history and development of CRISPR-Cas9 genome editing technology. It discusses how early approaches to targeted DNA cleavage relied on site-specific DNA recognition by oligonucleotides or proteins. Zinc finger nucleases and TAL effector nucleases were developed that allowed for genome editing but were difficult to design and validate. The discovery of CRISPR immune systems in bacteria provided the basis for a new approach using Cas9 guided by CRISPR RNA. Experiments showed Cas9 could be programmed to cleave DNA sequences using a simple RNA guide. This established the CRISPR-Cas9 system as an easy and efficient tool for genome editing that has enabled many new applications across biology.
Paired genes in stem cells shed new light on gene organization and regulation and epigenetic control of cardiogenesis. A study found that about 65% of long non-coding RNAs (lncRNAs) are transcribed in pairs with messenger RNAs (mRNAs) at active gene promoters in embryonic stem cells. Additionally, an experiment deactivated a lncRNA gene causing disruption in heart development and defects in mouse embryos, demonstrating that lncRNAs regulate tissue development and organogenesis. The discoveries provide new insights into gene transcription and the epigenetic roles of lncRNAs in development.
High-throughput RNA sequencing with Thermostable Group II Intron Reverse Tran...Douglas Wu
TGIRT is a thermostable group II intron reverse transcriptase that enables high-throughput RNA sequencing with several advantages over other reverse transcriptases. It recapitulates the relative abundance of transcripts, has higher strand-specificity, and provides more uniform gene coverage from 5' to 3' compared to TruSeq v3. TGIRT also enables simultaneous profiling of mRNA, lncRNA, and other structured small RNAs. Its applications include structured RNA-seq, plasma and exosomal RNA-seq for cancer diagnostics, and generation of long cDNAs.
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
This document describes a method for performing RNA sequencing on single nuclei. Key points:
1) The authors demonstrate that double-stranded cDNA can be synthesized from single mouse neural progenitor cell nuclei and hippocampal tissue nuclei, allowing for whole transcriptome sequencing.
2) On average, sequencing of single nuclei detected over 16,000 of the approximately 24,000 mouse protein-coding genes.
3) Analysis of single nuclei avoids issues with dissociating intact cells from complex tissues, is applicable across eukaryotic species, and provides insight into nuclear gene regulation.
4) RNA sequencing of single nuclei is a powerful new method for investigating gene expression at the single cell level without disrupting cells.
Use of Thermostable Group II Intron Reverse Transcriptases (TGIRTs) for Singl...Douglas Wu
(1) Thermostable group II intron reverse transcriptase (TGIRT) enzymes enable efficient single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA (cfDNA) fragments in human plasma and other bodily fluids. (2) ssDNA-seq using TGIRT can identify epigenetic features in cfDNA fragments that provide information about the tissue of origin, with potential diagnostic applications. (3) TGIRT-based ssDNA-seq allows effective library construction from small amounts of degraded cfDNA samples.
Use of Thermostable Group II Intron Reverse Transcriptases (TGIRTs) for Singl...Douglas Wu
Thermostable group II intron reverse transcriptases (TGIRTs) enable efficient single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA fragments in human plasma in 2 hours with fewer reagents. TGIRT-seq analysis of nucleosome positioning and transcription factor occupancy in plasma DNA matches previous studies and can determine tissue-of-origin. While TGIRT-seq has a higher mismatch rate than other methods, it produces comparable genomic coverage and can detect nucleosome positioning, transcription factor binding sites, and tissue-of-origin from plasma DNA.
Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.
Scientific research paper summary for class on molecular genetics. Original research on the development and use of long non-coding RNA in biological systems like CRISPR and the origin of that mechanism.
This document describes a comparative analysis of the human gut microbiota of Koreans using barcoded pyrosequencing. It finds that the Korean gut microbiome has high diversity at the species and strain levels, with over 800 species-level phylotypes identified on average per individual. The analysis identifies 14 core genera that are consistently present across Korean guts, including Bacteroides, Prevotella, Clostridium, and Ruminococcus. The phylum-level diversity of the Korean gut microbiome is similar to other human populations.
This study identified potentially plasmid sequences in strains of Micrococcus and Exiguobacterium bacteria. Contigs were generated from sequencing reads and analyzed with plasmid detection tools. For Micrococcus strains, 130 potential plasmid sequences were found, including 78 circular plasmids. Fewer potential sequences were identified in Exiguobacterium. Some sequences matched known plasmids, while others were novel. This analysis provides a basis for further studies of the plasmid content of these genera.
This document summarizes a presentation given by Minoru Kubo on single cell analyses for plant reprogramming studies. The presentation covered how next generation sequencing and single cell analyses have changed biological research. It discussed single cell transcriptomics for analyzing plant reprogramming at the single cell level. The goal is to identify reprogramming genes, understand cell-cell interactions during reprogramming, and validate theories of reprogramming using the moss Physcomitrella patens as a model organism. Positioning information from single cell analyses could provide new insights into cell lineages, interactions, and multicellular development.
The document discusses the longstanding rivalry between RNA and DNA, which are described as warring cousins. It suggests RNA was originally the dominant molecule involved in early life before DNA usurped its role. Now, RNA wages guerrilla warfare against DNA's supremacy through viruses. The internecine conflict between RNA and DNA plays out through continual mutations and spread of contagions within and between species. Possible outcomes of this warfare include one cousin achieving final dominance, a negotiated peace, or organisms developing independence from both molecules.
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
This document provides information on CRISPR Cas9 genome editing. It discusses the history and discovery of CRISPR dating back to 1987. It describes the key components of the CRISPR Cas9 system including Cas9 proteins, CRISPR RNA, protospacers, and PAM sequences. The mechanisms of how CRISPR Cas9 edits genomes through double strand breaks is explained. Finally, applications of CRISPR Cas9 are summarized, including using it to correct genetic mutations causing diseases in animals and potential applications in humans.
CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
- The document discusses research on the cotton root-knot nematode (Meloidogyne incognita), an endoparasitic nematode that infects thousands of plant species and causes billions in crop damage worldwide.
- The research uses Arabidopsis thaliana plants, including mutant strains, to study the nematode's infection process. Mutant plants have mutations in an ABC transporter gene (At4g15320) involved in secreting chemicals that nematodes use to locate host plants.
- The goal is to see if the mutant transporter gene affects nematode infection rates compared to wild-type Arabidopsis. Results found the mutant was actually not a true mutant and showed similar nematode infection to the
As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is
an immediate need for a simple, rapid, early, and sensitive point-of-care testing for COVID-
19 disease. Recently,
clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several
challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.
This document describes an experiment to test the effectiveness of different bacterial transformation mixes in CRISPR-Cas9 systems. The standard transformation mix contains calcium chloride while the experimental mix adds polyethylene glycol and dimethyl sulfoxide. Bacteria are made competent using each mix and transformed with CRISPR components before plating. Preliminary results showed more growth with the standard mix, but the experimental mix supported slightly more antibiotic-resistant growth. Multiple errors prevented conclusions, requiring further replication and controls.
This document discusses the CRISPR-Cas9 system for genome editing and its applications. It provides a brief history of CRISPR's discovery and development as a tool. CRISPR-Cas9 uses CRISPR sequences and Cas9 nuclease to precisely target and edit DNA. The document outlines the basic components and mechanisms of CRISPR-Cas9, as well as its current applications in cancer immunotherapy, inhibiting angiogenesis, and correcting genetic disorders.
This document provides an introduction to non-coding RNAs (ncRNAs). It discusses how RNA was proposed to have come before DNA and proteins in the RNA world hypothesis. It notes that RNA can store genetic information like DNA and have catalytic abilities like proteins. The document outlines different types and functions of ncRNAs, including how some act as guides for chemical modifications, regulate gene expression as sponges for small RNAs or proteins, or sense environmental signals as riboswitches or thermosensors. Examples of conserved ncRNA families and mechanisms are briefly described.
CRISPR-Cas9 Review: A potential tool for genome editingDavient Bala
The document discusses CRISPR-Cas9 as a potential tool for genome editing. It describes how CRISPR was originally discovered in bacteria and archaea as a mechanism for adaptive immunity against viruses. The CRISPR-Cas9 system uses guide RNA to direct an endonuclease called Cas9 to introduce targeted double-strand breaks in DNA, which can then be repaired through non-homologous end joining or homology directed repair for genome editing. Applications discussed include using CRISPR-Cas9 for disease modeling in animals and cell lines more efficiently compared to previous methods, as well as for drug development by generating gene knockouts and mutations for target validation.
1) The document discusses the CRISPR-Cas9 system of genome editing and its applications.
2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs.
3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.
CRISPR: what it is, and why it is having a profound impact on human healthPistoia Alliance
This document summarizes a webinar on CRISPR that included presentations from experts in gene editing and bioinformatics. The webinar provided an overview of CRISPR and how it works using the Cas9 enzyme and guide RNA to make precise cuts in DNA. It discussed how CRISPR is being used for gene knockout studies, clinical trials to treat diseases like cystic fibrosis and cancer, and the challenges of predicting off-target effects. The webinar highlighted both the promise and challenges of CRISPR for accelerating scientific discovery and developing new gene therapies.
Lessons learned from high throughput CRISPR targeting in human cell linesChris Thorne
In just a short period of time CRISPR-Cas9 technology has revolutionized the field of genome editing, and taken the scientific community by storm. Already our understanding of how best to apply this technology has advanced significantly and almost every week new publications appear showcasing its application in basic and translational research.
While CRISPR-Cas9 is applicable across many different cell types, we have found it particularly suited for genome editing in near-haploid human cell lines. This has allowed us to establish a robust pipeline for the inactivation of non-essential genes at unprecedented scale and efficiency.
We have now knocked out over 1500 human genes and have generated a resource that is, to the best of our knowledge, the largest collection of human knockout cell lines available, covering comprehensive subsets of genes clustered by biological pathway (e.g. the autophagy pathway, the JAK/STAT pathway) or by phylogenetic relationship (e.g. kinases, bromodomain-containing proteins).
In this talk we will discuss how, through more than 1500 genome editing experiments, we have started to unravel some of the general principles governing the use of CRISPR-Cas9 in mammalian cells. For example, we have analyzed the impact of variation in the guide RNA sequence on Cas9 cleavage efficiency and characterized the mutational signature arising from CRISPR-Cas9 cleavage.
We will also highlight (with examples) how these learnings are now being applied to introduce other genomic modifications in a high throughput manner, including chromosomal deletions, translocations, point mutations and endogenous gene tags.
Bio380 Human Evolution: Waking the deadMark Pallen
Bio380 Human Evolution, genes and genomes lecture on contribution of archaic populations to gene pool of anatomically modern humans, including Neanderthals and Denisovan
A SNP array for human population genetics studiesAffymetrix
Yontao Lu, Teri Genschoreck, Swapan Mallick, Amy Ollmann, Nick Patterson, Yiping Zhan, Teresa Webster, David Reich Overview of the Human Origins Array, the first array developed with leading geneticists to enable rigorous population genetics studies.
This document reanalyzes ancient mitochondrial DNA sequences recovered from Neandertal bones. Previous studies placed Neandertals at the base of the modern human phylogenetic tree, suggesting they did not contribute to the modern human gene pool. However, these analyses did not account for high substitution rate variation among sites in the human mitochondrial D-loop region or estimate nucleotide substitution model parameters. The authors reanalyze the Neandertal sequences using maximum likelihood methods that account for these factors to provide a more accurate phylogenetic reconstruction.
Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.
Scientific research paper summary for class on molecular genetics. Original research on the development and use of long non-coding RNA in biological systems like CRISPR and the origin of that mechanism.
This document describes a comparative analysis of the human gut microbiota of Koreans using barcoded pyrosequencing. It finds that the Korean gut microbiome has high diversity at the species and strain levels, with over 800 species-level phylotypes identified on average per individual. The analysis identifies 14 core genera that are consistently present across Korean guts, including Bacteroides, Prevotella, Clostridium, and Ruminococcus. The phylum-level diversity of the Korean gut microbiome is similar to other human populations.
This study identified potentially plasmid sequences in strains of Micrococcus and Exiguobacterium bacteria. Contigs were generated from sequencing reads and analyzed with plasmid detection tools. For Micrococcus strains, 130 potential plasmid sequences were found, including 78 circular plasmids. Fewer potential sequences were identified in Exiguobacterium. Some sequences matched known plasmids, while others were novel. This analysis provides a basis for further studies of the plasmid content of these genera.
This document summarizes a presentation given by Minoru Kubo on single cell analyses for plant reprogramming studies. The presentation covered how next generation sequencing and single cell analyses have changed biological research. It discussed single cell transcriptomics for analyzing plant reprogramming at the single cell level. The goal is to identify reprogramming genes, understand cell-cell interactions during reprogramming, and validate theories of reprogramming using the moss Physcomitrella patens as a model organism. Positioning information from single cell analyses could provide new insights into cell lineages, interactions, and multicellular development.
The document discusses the longstanding rivalry between RNA and DNA, which are described as warring cousins. It suggests RNA was originally the dominant molecule involved in early life before DNA usurped its role. Now, RNA wages guerrilla warfare against DNA's supremacy through viruses. The internecine conflict between RNA and DNA plays out through continual mutations and spread of contagions within and between species. Possible outcomes of this warfare include one cousin achieving final dominance, a negotiated peace, or organisms developing independence from both molecules.
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
This document provides information on CRISPR Cas9 genome editing. It discusses the history and discovery of CRISPR dating back to 1987. It describes the key components of the CRISPR Cas9 system including Cas9 proteins, CRISPR RNA, protospacers, and PAM sequences. The mechanisms of how CRISPR Cas9 edits genomes through double strand breaks is explained. Finally, applications of CRISPR Cas9 are summarized, including using it to correct genetic mutations causing diseases in animals and potential applications in humans.
CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
- The document discusses research on the cotton root-knot nematode (Meloidogyne incognita), an endoparasitic nematode that infects thousands of plant species and causes billions in crop damage worldwide.
- The research uses Arabidopsis thaliana plants, including mutant strains, to study the nematode's infection process. Mutant plants have mutations in an ABC transporter gene (At4g15320) involved in secreting chemicals that nematodes use to locate host plants.
- The goal is to see if the mutant transporter gene affects nematode infection rates compared to wild-type Arabidopsis. Results found the mutant was actually not a true mutant and showed similar nematode infection to the
As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is
an immediate need for a simple, rapid, early, and sensitive point-of-care testing for COVID-
19 disease. Recently,
clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several
challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.
This document describes an experiment to test the effectiveness of different bacterial transformation mixes in CRISPR-Cas9 systems. The standard transformation mix contains calcium chloride while the experimental mix adds polyethylene glycol and dimethyl sulfoxide. Bacteria are made competent using each mix and transformed with CRISPR components before plating. Preliminary results showed more growth with the standard mix, but the experimental mix supported slightly more antibiotic-resistant growth. Multiple errors prevented conclusions, requiring further replication and controls.
This document discusses the CRISPR-Cas9 system for genome editing and its applications. It provides a brief history of CRISPR's discovery and development as a tool. CRISPR-Cas9 uses CRISPR sequences and Cas9 nuclease to precisely target and edit DNA. The document outlines the basic components and mechanisms of CRISPR-Cas9, as well as its current applications in cancer immunotherapy, inhibiting angiogenesis, and correcting genetic disorders.
This document provides an introduction to non-coding RNAs (ncRNAs). It discusses how RNA was proposed to have come before DNA and proteins in the RNA world hypothesis. It notes that RNA can store genetic information like DNA and have catalytic abilities like proteins. The document outlines different types and functions of ncRNAs, including how some act as guides for chemical modifications, regulate gene expression as sponges for small RNAs or proteins, or sense environmental signals as riboswitches or thermosensors. Examples of conserved ncRNA families and mechanisms are briefly described.
CRISPR-Cas9 Review: A potential tool for genome editingDavient Bala
The document discusses CRISPR-Cas9 as a potential tool for genome editing. It describes how CRISPR was originally discovered in bacteria and archaea as a mechanism for adaptive immunity against viruses. The CRISPR-Cas9 system uses guide RNA to direct an endonuclease called Cas9 to introduce targeted double-strand breaks in DNA, which can then be repaired through non-homologous end joining or homology directed repair for genome editing. Applications discussed include using CRISPR-Cas9 for disease modeling in animals and cell lines more efficiently compared to previous methods, as well as for drug development by generating gene knockouts and mutations for target validation.
1) The document discusses the CRISPR-Cas9 system of genome editing and its applications.
2) CRISPR-Cas9 allows for accurate and multiplex gene modification guided by RNA and is an advanced technique compared to earlier tools like ZFNs and TALENs.
3) The document covers the mechanism of CRISPR-Cas9 immunity in bacteria, the general protocol for genome editing using CRISPR-Cas9, and new developments like modified Cas9 enzymes and the Cpf1 protein.
CRISPR: what it is, and why it is having a profound impact on human healthPistoia Alliance
This document summarizes a webinar on CRISPR that included presentations from experts in gene editing and bioinformatics. The webinar provided an overview of CRISPR and how it works using the Cas9 enzyme and guide RNA to make precise cuts in DNA. It discussed how CRISPR is being used for gene knockout studies, clinical trials to treat diseases like cystic fibrosis and cancer, and the challenges of predicting off-target effects. The webinar highlighted both the promise and challenges of CRISPR for accelerating scientific discovery and developing new gene therapies.
Lessons learned from high throughput CRISPR targeting in human cell linesChris Thorne
In just a short period of time CRISPR-Cas9 technology has revolutionized the field of genome editing, and taken the scientific community by storm. Already our understanding of how best to apply this technology has advanced significantly and almost every week new publications appear showcasing its application in basic and translational research.
While CRISPR-Cas9 is applicable across many different cell types, we have found it particularly suited for genome editing in near-haploid human cell lines. This has allowed us to establish a robust pipeline for the inactivation of non-essential genes at unprecedented scale and efficiency.
We have now knocked out over 1500 human genes and have generated a resource that is, to the best of our knowledge, the largest collection of human knockout cell lines available, covering comprehensive subsets of genes clustered by biological pathway (e.g. the autophagy pathway, the JAK/STAT pathway) or by phylogenetic relationship (e.g. kinases, bromodomain-containing proteins).
In this talk we will discuss how, through more than 1500 genome editing experiments, we have started to unravel some of the general principles governing the use of CRISPR-Cas9 in mammalian cells. For example, we have analyzed the impact of variation in the guide RNA sequence on Cas9 cleavage efficiency and characterized the mutational signature arising from CRISPR-Cas9 cleavage.
We will also highlight (with examples) how these learnings are now being applied to introduce other genomic modifications in a high throughput manner, including chromosomal deletions, translocations, point mutations and endogenous gene tags.
Bio380 Human Evolution: Waking the deadMark Pallen
Bio380 Human Evolution, genes and genomes lecture on contribution of archaic populations to gene pool of anatomically modern humans, including Neanderthals and Denisovan
A SNP array for human population genetics studiesAffymetrix
Yontao Lu, Teri Genschoreck, Swapan Mallick, Amy Ollmann, Nick Patterson, Yiping Zhan, Teresa Webster, David Reich Overview of the Human Origins Array, the first array developed with leading geneticists to enable rigorous population genetics studies.
This document reanalyzes ancient mitochondrial DNA sequences recovered from Neandertal bones. Previous studies placed Neandertals at the base of the modern human phylogenetic tree, suggesting they did not contribute to the modern human gene pool. However, these analyses did not account for high substitution rate variation among sites in the human mitochondrial D-loop region or estimate nucleotide substitution model parameters. The authors reanalyze the Neandertal sequences using maximum likelihood methods that account for these factors to provide a more accurate phylogenetic reconstruction.
Masters dissertation work - Human genome diversity Pradeep B J Reddy
This document discusses molecular biology research into human genetic diversity and evolution. It focuses on analyzing mitochondrial DNA from different populations to understand genetic relationships and test hypotheses about human origin. The research project aims to study genomic diversity within and between endogamous populations in India by sequencing mitochondrial DNA and analyzing the data to construct evolutionary networks and trees. Over 6,000 blood samples from various populations have been analyzed.
Close correspondence between quantitative and molecular-genetic divergence t...Kristian Pedersen
This document presents a new method for estimating divergence times between populations based on quantitative traits like cranial measurements. The authors develop a phenotypic divergence time (PTD) estimator modeled after genetic divergence time estimators. They apply their PTD estimator to cranial measurements from over 2,500 modern humans and 20 Neandertals. Their analysis estimates the Neandertal-modern human divergence at either 311,000 years ago or 435,000 years ago, depending on assumptions. These dates are similar to estimates from ancient DNA, implying cranial and genetic divergence largely track population history through neutral evolution.
A consortium of 440 scientists from 32 laboratories characterized functional elements in the human genome as part of the ENCyclopedia Of DNA Elements (ENCODE) project. They found that 80% of the genome is biochemically active, with millions of regulatory elements such as promoters, enhancers, and insulators. Many of these elements interact with genes over long distances to control gene expression. This study significantly changes understanding of how the genome works.
DNA probes and PCR technology are used to rapidly identify microorganisms by amplifying copies of their DNA or RNA. The amplified DNA can then be detected and quantified to assess how many microorganisms are present and monitor responses to treatment. DNA technology can also break the cycle of disease in nature by intervening in parasites' life cycles. DNA barcoding uses a short, unique DNA sequence to identify species, including establishing new species. It supplements traditional taxonomy. Recovering DNA from fossils through PCR amplification allows comparison to present DNA, determining relationships and identifying ancestors. Tracking human mtDNA and Y chromosomes shows all people originated from an African ancestor. Cloning extinct animals could disrupt nature but may be acceptable for those driven extinct by humans to preserve
Detecting ancient admixture in humans using sequence polymorphism data (wall)Kristian Pedersen
This document summarizes a study that examines how much genetic data would be needed to determine if archaic human populations like Neanderthals interbred with and contributed to the modern human gene pool. The study introduces a model of ancient population structure involving mixing between groups. It then determines how many unlinked nuclear loci from sequencing data would be required to distinguish between scenarios of single origin vs. multiregional origin of modern humans. The study finds that 50-100 loci would be needed if plausible parameter estimates are used, and that the amount of available data is not yet sufficient to support one model over the other. However, with more sequencing in coming years, the required data should become available to help resolve the debate.
An Evolutionarily Conserved Long Noncoding RNA TUNA Controls Pluripotency and...Zach Rana
Here are 3 sentences summarizing the key points of the document:
1) An unbiased genome-scale RNAi screen in mouse embryonic stem cells identified 20 long noncoding RNAs (lincRNAs) required for maintenance of pluripotency, including linc86023 (also known as TUNA or megamind).
2) TUNA was found to form a complex with RNA-binding proteins at the promoters of pluripotency genes Nanog, Sox2, and Fgf4, and its depletion inhibited neural differentiation and impaired proliferation of mouse ESCs.
3) TUNA showed evolutionary conservation and central nervous system-restricted expression in vertebrates, and its knockdown
The human genome project aimed to determine the complete DNA sequence of humans. It began in 1990 and was declared complete in 2003. The goals were to optimize data analysis, sequence the entire genome, and identify all human genes. Scientists isolated DNA from cells, broke it into fragments, cloned the fragments into hosts, and used Sanger sequencing to determine the sequence and arrange fragments into chromosomes. The project found that humans have around 30,000 genes, less than previously thought, and junk DNA makes up much of the genome. It has advanced disease research and treatments.
Basic concepts & scope of recombinant DNA technologyRavi Kant Agrawal
Recombinant DNA technology involves combining DNA molecules from different sources and introducing them into host cells. Key developments include the discovery that DNA carries genetic information (Avery, 1944), determining DNA's structure (Watson and Crick, 1953), developing techniques to cut and join DNA (restriction enzymes and ligase, 1970s), and creating the first recombinant DNA molecules by combining bacterial plasmid and phage DNA (Cohen and Boyer, 1973). These advances laid the foundation for genetic engineering.
Genome editing methods such as ZFNs, TALENs, and CRISPR/Cas9 use engineered nucleases to create targeted double-stranded breaks in DNA which are then repaired through endogenous cellular processes. These nucleases can be used to modify genomes through techniques like gene knockout, targeted mutation insertion/deletion/correction, and studying gene function. CRISPR/Cas9 uses a guide RNA and Cas9 nuclease to target specific DNA sequences for editing. The four main steps for CRISPR are: 1) selecting target sequences near a PAM site, 2) designing and cloning gRNA, 3) delivering Cas9 and gRNA into cells, and 4) DNA repair after cleavage results in gene modification
This document summarizes an experiment aiming to knockout the CTNNB1 gene in mouse embryonic stem cells using CRISPR-Cas9 genome editing. It describes designing a guide RNA targeting exon 10 of CTNNB1, cloning it into a vector, transforming E. coli, and testing primers for detecting knockout via PCR and qPCR. Prior studies showed CTNNB1 knockout embryos had defects in ectoderm and mesoderm development. The authors hypothesized knocking out CTNNB1 in stem cells would produce inviable embryos, similar to prior findings.
The document summarizes the Human Genome Project (HGP). It began in 1990 with the goal of identifying all the genes in human DNA and determining the sequence of the 3 billion chemical base pairs. The 13-year project was completed in 2003 and involved international collaboration. It mapped the human genome and identified approximately 20,000-25,000 human genes. The HGP provided insights into human evolution and has applications in medicine, such as for identifying genes associated with diseases. It also advanced bioinformatics for analyzing large DNA datasets.
This document discusses the polymerase chain reaction (PCR) amplification of the Alu sequence, a repetitive element in the human genome, from DNA samples. The author describes extracting DNA from cheek cells using a buccal swab and saline rinse, then using PCR to amplify the Alu sequence region. After PCR, the amplified DNA fragments will be run on an agarose gel electrophoresis to check for the presence of the Alu sequence band. The results will then be compared between samples from the author's biology lab class and published data from the United States.
- DNA was first isolated and characterized in 1868, and in 1944 it was discovered that DNA carries genetic information. Experiments in 1952 confirmed that DNA, not protein, carries genetic material.
- Rosalind Franklin and Maurice Wilkins used X-ray diffraction in the early 1950s to show that DNA has a helical structure with periodicities of 3.4Å and 34Å.
- Watson and Crick proposed in 1953 that DNA consists of two antiparallel chains in a right-handed double helix, with complementary base pairs (A=T, G=C) forming hydrogen bonds between the chains. This model explained DNA replication through template-directed synthesis of new strands.
The Human Genome Project was an international scientific research project with the goal of determining the sequence of nucleotide base pairs that make up human DNA. It originally aimed to map the over three billion nucleotides contained in the human genome. The finished human genome is a mosaic assembled from sequencing a small number of individuals. The project has provided insights into human genetics and disease research.
The document discusses several topics related to molecular biology including:
1) It provides an overview of the central dogma of biology - how DNA is transcribed into RNA and then translated into proteins in both bacterial and eukaryotic cells.
2) It lists some uses of molecular biology techniques in medicine, agriculture, industry, forensics, and other fields like diagnosing diseases, developing pest-resistant crops, and DNA fingerprinting.
3) It briefly describes the history and process of polymerase chain reaction (PCR), including how thermal cyclers allow many reactions to be run simultaneously, accelerating results from days to hours.
The human genome project was started in 1990 with the goal of sequencing and ...Rania Malik
The Human Genome Project was a 13-year effort that began in 1990 with the goal of sequencing the entire human genome and identifying all of the genes in order to better understand genetic diseases and develop new treatments. It involved mapping the genome by identifying genetic markers and then sequencing DNA using techniques like cloning and polymerase chain reaction to amplify DNA for analysis. The project was completed in 2003, earlier than expected due to advances in sequencing technology, providing a full sequence of the human genome.
Similar to Dna sequence of the mitochondrial hypervariable region ii (krings et al.) (20)
The document discusses the religious beliefs and practices in Scandinavia from 600-900 AD. It describes the principal deities belonging to the Æsir and Vanir groups, which were sometimes in conflict but later reconciled. The Æsir such as Odin and Thor were more popular in western Scandinavia, while the Vanir like Njord and Freyr were predominant in the east. Lesser spiritual entities also played roles in rituals and beliefs. The text examines the distribution of cults across regions and outlines some of the major deities and myths from Old Norse sources.
The document discusses the social structure and political institutions of Scandinavian communities during the Viking Age from 700-900 AD. It analyzes the social hierarchy and divisions based on sex, age, and status using archaeological evidence, textual sources, mythical tales, and literary accounts. Women generally had high social status and equal rights to men. The sources describe distinct social classes of slaves, freemen, and rulers. Archaeological evidence like the rich Oseberg ship burial shows some women held elite aristocratic roles. Overall, the document aims to understand Viking society by examining evidence from multiple genres of sources.
The document discusses the origins of Viking culture in Scandinavia during the Bronze Age and Iron Age. It describes how aspects of culture developed over this period, including ship building, domestic architecture like longhouses, religious practices, and economic activities like agriculture, fishing, and trade. Society was stratified with evidence of wealthier individuals. Overall, the foundations for the expansion of the Viking Age were established through cultural developments spanning many centuries prior.
This document provides an overview of a course on the Vikings between AD 700-1100. It discusses the sources used to study the Vikings, including historical accounts, sagas, runic inscriptions and place names. It also outlines advances in archaeological techniques that have improved understanding of Viking sites, settlements, trade and environment. Interpretive paradigms have shifted from seeing Vikings as pirates to appreciating their social structure and role in state formation, trade and assimilation into other cultures.
Anterior tooth growth periods in neandertals were comparable to those of mode...Kristian Pedersen
This document analyzes the duration of enamel formation in anterior teeth of Neanderthals compared to three modern human groups. It finds that while Neanderthal formation times were likely faster than Inuit, they were not faster than those of an English sample and were clearly slower than a southern African sample. This suggests Neanderthal tooth growth, and by extension somatic growth, falls within the range of modern human variation.
A re appraisal of ceprano calvaria affinities (ascenzi et al.)Kristian Pedersen
The document discusses new modifications made to the reconstruction of the Ceprano calvaria based on additional examination. It provides a reassessment of the morphology and measurements of the calvaria and evaluates its affinities to Homo erectus. New geological data from the Priverno Basin suggests an age of 800-900 thousand years for the calvaria. While showing some similarities, the calvaria exhibits significant morphological variation compared to typical H. erectus specimens.
A modern human pattern of dental development in lower pleistocene hominids fr...Kristian Pedersen
This document summarizes a study of dental development patterns in hominid fossils from the Gran Dolina site in Spain. The fossils are dated to around 0.8 million years ago. Three individuals provided information about their dental development:
1) Hominid 1 showed signs of stress during childhood in dental enamel and dentine.
2) Hominid 2 and 3 died during early childhood before their third molars erupted.
3) Analysis of tooth mineralization stages in the fossils showed more similarity to patterns in modern humans than chimpanzees. This supports the view that by 0.8 million years ago, Homo species had a prolonged developmental pattern like humans.
A cranium for the earliest europeans: phylogenetic position of the hominid fr...Kristian Pedersen
The document summarizes a study analyzing the cranium of a hominid fossil discovered near Ceprano, Italy in 1994. Researchers estimated its age to be between 800-900 thousand years old based on dating of surrounding rock layers. The cranium was reconstructed and compared morphologically to other early European and Asian hominin fossils. Results found it displayed a unique blend of traits linking early Homo erectus to later specimens referred to Homo heidelbergensis. The researchers concluded the Ceprano cranium represents an important transitional form between early hominin groups, filling a gap in the human fossil record in Europe between 1-0.5 million years ago.
A calvarium of late homo erectus from ceprano, italy (ascenzi et al.)Kristian Pedersen
The document summarizes the discovery and context of a late Homo erectus calvarium found in Ceprano, Italy. Key points:
- The calvarium was discovered in 1994 during highway construction in a clay layer dated to around 700,000 years ago.
- The shape and size of the calvarium (capacity of 1185 ml) show it possesses some, but not all, features of Homo erectus.
- A geological survey found the remains in a colluvial-alluvial paleosol originating from an ancient slope, indicating the site was a marshy pool within the Ceprano basin.
The feeding biomechanics and dietary ecology of australopithecus africanus (s...Kristian Pedersen
1) The feeding biomechanics and craniofacial morphology of Australopithecus africanus were analyzed using finite element analysis informed by comparative data.
2) The results suggest that the facial skeleton of A. africanus was well-adapted to withstand loads applied during premolar biting, including stresses along the nasal margins and zygomatic root.
3) However, neither the mastication of small hard objects nor large volumes of food alone can fully explain the evolution of the robust facial features in A. africanus. Rather, key aspects are more likely related to ingesting and processing large mechanically protected foods like nuts and seeds.
The carbon isotope ecology and diet of australopithecus africanus at sterkfon...Kristian Pedersen
The document summarizes a study that analyzed the carbon isotope ratios in tooth enamel from 10 specimens of Australopithecus africanus from Sterkfontein in South Africa, dating to between 2.5-2.0 million years ago. The results show that A. africanus had a varied diet incorporating both C3 forest foods and C4 savanna foods to a greater degree than other early hominins. This suggests A. africanus was a highly adaptable and opportunistic feeder. The isotope data also indicates the individuals exhibited more dietary variation than other early hominin species, arguing against suggestions that multiple species are represented in the A. africanus taxon.
Ontogeny of australopithecines and early homo: evidence from carnial capacity...Kristian Pedersen
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive function. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
Cosmogenic Nuclide Dating Of Sahelanthropus Tchadensis And Australopiethcus B...Kristian Pedersen
This study used cosmogenic nuclide dating to determine the ages of sedimentary units containing hominid remains at two localities in Chad - KT 12 locality in Koro Toro and TM 266 locality in Toros-Menalla. At KT 12, which contained Australopithecus bahrelghazali, authigenic 10Be/9Be dating of the sedimentary level yielded an age of 3.58 ± 0.27 Ma, consistent with an early Pliocene age estimation. At TM 266, where Sahelanthropus tchadensis was found, 28 10Be/9Be ages from the anthracotheriid unit containing the hominid remains bracketed its age between 6.8
The document discusses the emergence and characteristics of Neanderthals. Some key points:
- Neanderthals first emerged in Europe and the Middle East between 150,000-200,000 years ago as a distinct species from Homo heidelbergensis.
- They were restricted to Europe and Western Asia, with no evidence found in Africa or East Asia.
- Anatomically, Neanderthals had stockier builds than modern humans and more robust skeletons, especially in the cranium.
- Genetic evidence shows Neanderthals diverged from the modern human line well before 300,000 years ago, with a substantial genetic distance between the two groups.
-
Homo erectus expanded out of Africa into Eurasia around 1.6 million years ago. Some of the earliest sites outside of Africa are found in East Asia, particularly China and Java, dating back to around 1.9-1.7 million years ago. Homo erectus inhabited sites across Asia utilizing chopper tool industries rather than hand-axes. Some of the most important early Asian sites include Longgupo Cave in China dating to 1.7-1.9 million years ago, and Sangiran in Java dating to around 1.6 million years ago. Homo erectus expanded into Europe around 1 million years ago, with some of the earliest securely dated sites found in Spain and Italy.
Homo ergaster was an early form of Homo that expanded out of Africa between 2-1.6 million years ago. Evidence from the Dmanisi site in Georgia dates Homo ergaster occupation there to 1.88 million years ago, indicating they dispersed earlier in Africa. Homo ergaster displayed increased brain size and cranial capacity compared to Homo habilis, and were taller yet more gracile. They controlled fire, made Acheulean stone tools like handaxes, and were the first hominins to colonize areas outside of Africa, suggesting increased social complexity and technological adaptation.
This document discusses early hominin species including Australopithecus garhi, Homo habilis, and Homo rudolfensis. A. garhi, found in Ethiopia in 1996, was the earliest known tool-producing hominin dating to between 2.6-2.5 million years ago. H. habilis produced basic stone tools and expanded out of Africa between 2.4-1.5 million years ago. H. rudolfensis, found in Kenya, may be a separate species from H. habilis based on cranial differences. The document also examines the physical characteristics, fossil evidence, locomotion, diet, and basic stone tool production of these early hominins.
The document summarizes key australopithecine species and their evolution. It discusses Australopithecus anamensis, A. afarensis, A. africanus, Paranthropus aethiopicus, P. boisei, P. robustus, and A. garhi. It describes their fossil records, anatomies, habitats, locomotion, and relationships. A. garhi is significant as the earliest known tool-making hominin. Debate continues regarding the lineages and relationships between the gracile and robust australopithecine forms.
The document discusses evidence from genetics, anatomy, and behavior that traces the evolutionary path from early primates to Homo sapiens. It describes several early primate species such as Proconsul and Sivapithecus found in Africa and Asia dating back millions of years. Genetic evidence shows humans share over 99% of DNA with chimpanzees and diverged from them around 5-6 million years ago. Anatomical similarities and differences in the pelvis, feet and locomotion provide insights into the emergence of bipedalism. Higher primates display sophisticated communication including gestures, facial expressions, and tool usage, providing clues about early human behavior.
This document provides an introduction to the study of human evolution, including historical perspectives and current issues. It discusses how evolutionary theory conflictsed with religious traditions that view humans as separate from nature. It also outlines problems with the fossil record and reconciling evidence from different disciplines. Previously, religious traditions viewed humans as superior to other species in a "great chain of being." The biblical chronology of the earth's age was challenged by emerging geology in the 18th century, providing sufficient time for evolution to occur.
How information systems are built or acquired puts information, which is what they should be about, in a secondary place. Our language adapted accordingly, and we no longer talk about information systems but applications. Applications evolved in a way to break data into diverse fragments, tightly coupled with applications and expensive to integrate. The result is technical debt, which is re-paid by taking even bigger "loans", resulting in an ever-increasing technical debt. Software engineering and procurement practices work in sync with market forces to maintain this trend. This talk demonstrates how natural this situation is. The question is: can something be done to reverse the trend?
HCL Notes and Domino License Cost Reduction in the World of DLAUpanagenda
Webinar Recording: https://www.panagenda.com/webinars/hcl-notes-and-domino-license-cost-reduction-in-the-world-of-dlau/
The introduction of DLAU and the CCB & CCX licensing model caused quite a stir in the HCL community. As a Notes and Domino customer, you may have faced challenges with unexpected user counts and license costs. You probably have questions on how this new licensing approach works and how to benefit from it. Most importantly, you likely have budget constraints and want to save money where possible. Don’t worry, we can help with all of this!
We’ll show you how to fix common misconfigurations that cause higher-than-expected user counts, and how to identify accounts which you can deactivate to save money. There are also frequent patterns that can cause unnecessary cost, like using a person document instead of a mail-in for shared mailboxes. We’ll provide examples and solutions for those as well. And naturally we’ll explain the new licensing model.
Join HCL Ambassador Marc Thomas in this webinar with a special guest appearance from Franz Walder. It will give you the tools and know-how to stay on top of what is going on with Domino licensing. You will be able lower your cost through an optimized configuration and keep it low going forward.
These topics will be covered
- Reducing license cost by finding and fixing misconfigurations and superfluous accounts
- How do CCB and CCX licenses really work?
- Understanding the DLAU tool and how to best utilize it
- Tips for common problem areas, like team mailboxes, functional/test users, etc
- Practical examples and best practices to implement right away
Northern Engraving | Nameplate Manufacturing Process - 2024Northern Engraving
Manufacturing custom quality metal nameplates and badges involves several standard operations. Processes include sheet prep, lithography, screening, coating, punch press and inspection. All decoration is completed in the flat sheet with adhesive and tooling operations following. The possibilities for creating unique durable nameplates are endless. How will you create your brand identity? We can help!
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdfChart Kalyan
A Mix Chart displays historical data of numbers in a graphical or tabular form. The Kalyan Rajdhani Mix Chart specifically shows the results of a sequence of numbers over different periods.
For the full video of this presentation, please visit: https://www.edge-ai-vision.com/2024/06/temporal-event-neural-networks-a-more-efficient-alternative-to-the-transformer-a-presentation-from-brainchip/
Chris Jones, Director of Product Management at BrainChip , presents the “Temporal Event Neural Networks: A More Efficient Alternative to the Transformer” tutorial at the May 2024 Embedded Vision Summit.
The expansion of AI services necessitates enhanced computational capabilities on edge devices. Temporal Event Neural Networks (TENNs), developed by BrainChip, represent a novel and highly efficient state-space network. TENNs demonstrate exceptional proficiency in handling multi-dimensional streaming data, facilitating advancements in object detection, action recognition, speech enhancement and language model/sequence generation. Through the utilization of polynomial-based continuous convolutions, TENNs streamline models, expedite training processes and significantly diminish memory requirements, achieving notable reductions of up to 50x in parameters and 5,000x in energy consumption compared to prevailing methodologies like transformers.
Integration with BrainChip’s Akida neuromorphic hardware IP further enhances TENNs’ capabilities, enabling the realization of highly capable, portable and passively cooled edge devices. This presentation delves into the technical innovations underlying TENNs, presents real-world benchmarks, and elucidates how this cutting-edge approach is positioned to revolutionize edge AI across diverse applications.
Programming Foundation Models with DSPy - Meetup SlidesZilliz
Prompting language models is hard, while programming language models is easy. In this talk, I will discuss the state-of-the-art framework DSPy for programming foundation models with its powerful optimizers and runtime constraint system.
Your One-Stop Shop for Python Success: Top 10 US Python Development Providersakankshawande
Simplify your search for a reliable Python development partner! This list presents the top 10 trusted US providers offering comprehensive Python development services, ensuring your project's success from conception to completion.
Fueling AI with Great Data with Airbyte WebinarZilliz
This talk will focus on how to collect data from a variety of sources, leveraging this data for RAG and other GenAI use cases, and finally charting your course to productionalization.
Essentials of Automations: Exploring Attributes & Automation ParametersSafe Software
Building automations in FME Flow can save time, money, and help businesses scale by eliminating data silos and providing data to stakeholders in real-time. One essential component to orchestrating complex automations is the use of attributes & automation parameters (both formerly known as “keys”). In fact, it’s unlikely you’ll ever build an Automation without using these components, but what exactly are they?
Attributes & automation parameters enable the automation author to pass data values from one automation component to the next. During this webinar, our FME Flow Specialists will cover leveraging the three types of these output attributes & parameters in FME Flow: Event, Custom, and Automation. As a bonus, they’ll also be making use of the Split-Merge Block functionality.
You’ll leave this webinar with a better understanding of how to maximize the potential of automations by making use of attributes & automation parameters, with the ultimate goal of setting your enterprise integration workflows up on autopilot.
Introduction of Cybersecurity with OSS at Code Europe 2024Hiroshi SHIBATA
I develop the Ruby programming language, RubyGems, and Bundler, which are package managers for Ruby. Today, I will introduce how to enhance the security of your application using open-source software (OSS) examples from Ruby and RubyGems.
The first topic is CVE (Common Vulnerabilities and Exposures). I have published CVEs many times. But what exactly is a CVE? I'll provide a basic understanding of CVEs and explain how to detect and handle vulnerabilities in OSS.
Next, let's discuss package managers. Package managers play a critical role in the OSS ecosystem. I'll explain how to manage library dependencies in your application.
I'll share insights into how the Ruby and RubyGems core team works to keep our ecosystem safe. By the end of this talk, you'll have a better understanding of how to safeguard your code.
Discover top-tier mobile app development services, offering innovative solutions for iOS and Android. Enhance your business with custom, user-friendly mobile applications.
Ivanti’s Patch Tuesday breakdown goes beyond patching your applications and brings you the intelligence and guidance needed to prioritize where to focus your attention first. Catch early analysis on our Ivanti blog, then join industry expert Chris Goettl for the Patch Tuesday Webinar Event. There we’ll do a deep dive into each of the bulletins and give guidance on the risks associated with the newly-identified vulnerabilities.
Monitoring and Managing Anomaly Detection on OpenShift.pdfTosin Akinosho
Monitoring and Managing Anomaly Detection on OpenShift
Overview
Dive into the world of anomaly detection on edge devices with our comprehensive hands-on tutorial. This SlideShare presentation will guide you through the entire process, from data collection and model training to edge deployment and real-time monitoring. Perfect for those looking to implement robust anomaly detection systems on resource-constrained IoT/edge devices.
Key Topics Covered
1. Introduction to Anomaly Detection
- Understand the fundamentals of anomaly detection and its importance in identifying unusual behavior or failures in systems.
2. Understanding Edge (IoT)
- Learn about edge computing and IoT, and how they enable real-time data processing and decision-making at the source.
3. What is ArgoCD?
- Discover ArgoCD, a declarative, GitOps continuous delivery tool for Kubernetes, and its role in deploying applications on edge devices.
4. Deployment Using ArgoCD for Edge Devices
- Step-by-step guide on deploying anomaly detection models on edge devices using ArgoCD.
5. Introduction to Apache Kafka and S3
- Explore Apache Kafka for real-time data streaming and Amazon S3 for scalable storage solutions.
6. Viewing Kafka Messages in the Data Lake
- Learn how to view and analyze Kafka messages stored in a data lake for better insights.
7. What is Prometheus?
- Get to know Prometheus, an open-source monitoring and alerting toolkit, and its application in monitoring edge devices.
8. Monitoring Application Metrics with Prometheus
- Detailed instructions on setting up Prometheus to monitor the performance and health of your anomaly detection system.
9. What is Camel K?
- Introduction to Camel K, a lightweight integration framework built on Apache Camel, designed for Kubernetes.
10. Configuring Camel K Integrations for Data Pipelines
- Learn how to configure Camel K for seamless data pipeline integrations in your anomaly detection workflow.
11. What is a Jupyter Notebook?
- Overview of Jupyter Notebooks, an open-source web application for creating and sharing documents with live code, equations, visualizations, and narrative text.
12. Jupyter Notebooks with Code Examples
- Hands-on examples and code snippets in Jupyter Notebooks to help you implement and test anomaly detection models.
Monitoring and Managing Anomaly Detection on OpenShift.pdf
Dna sequence of the mitochondrial hypervariable region ii (krings et al.)
1. DNA sequence of the mitochondrial hypervariable region II from the Neandertal type
specimen
Matthias Krings, Helga Geisert, Ralf W. Schmitz, Heike Krainitzki, and Svante Pääbo
PNAS 1999;96;5581-5585
doi:10.1073/pnas.96.10.5581
This information is current as of January 2007.
Online Information High-resolution figures, a citation map, links to PubMed and Google
& Services Scholar, etc., can be found at:
www.pnas.org/cgi/content/full/96/10/5581
References This article cites 20 articles, 10 of which you can access for free at:
www.pnas.org/cgi/content/full/96/10/5581#BIBL
This article has been cited by other articles:
www.pnas.org/cgi/content/full/96/10/5581#otherarticles
E-mail Alerts Receive free email alerts when new articles cite this article - sign up in the
box at the top right corner of the article or click here.
Rights & Permissions To reproduce this article in part (figures, tables) or in entirety, see:
www.pnas.org/misc/rightperm.shtml
Reprints To order reprints, see:
www.pnas.org/misc/reprints.shtml
Notes:
2. Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 5581–5585, May 1999
Evolution
DNA sequence of the mitochondrial hypervariable region II from
the Neandertal type specimen
MATTHIAS K RINGS*, HELGA GEISERT*, RALF W. SCHMITZ†, HEIKE K RAINITZKI‡, AND SVANTE PAABO*§
¨¨
*Max Planck Institute for Evolutionary Anthropology, Inselstrasse 22, D-04103 Leipzig, Germany; †Landschaftsverband Rheinland, Rheinisches Amt fur
¨
Bodendenkmalpflege Rheinisches Landesmuseum, Bonn, Endenicher Strasse 133, D-53115 Bonn, Germany; and ‡Hohere Berufsfachschule fur
¨ ¨
praparationstechnische Assistenten, Markstrasse 185, D-44799 Bochum, Germany
¨
Edited by L. L. Cavalli-Sforza, Stanford University School of Medicine, Stanford, CA, and approved February 17, 1999 (received for review
December 28, 1998)
ABSTRACT The DNA sequence of the second hypervari- lyzed the relationship of the combined sequences to the
able region of the mitochondrial control region of the Nean- contemporary human mtDNA gene pool.
dertal type specimen, found in 1856 in central Europe, has
been determined from 92 clones derived from eight overlap- MATERIALS AND METHODS
ping amplifications performed from four independent ex-
tracts. When the reconstructed sequence is analyzed together Experimental Procedures. Sampling, precautions against
with the previously determined DNA sequence from the first contamination, DNA extraction, PCR amplifications, cloning
hypervariable region, the Neandertal mtDNA is found to fall of PCR products, and sequencing of clones were performed as
outside a phylogenetic tree relating the mtDNAs of contem- described (5). Extracts A, B, and C were prepared previously
porary humans. The date of divergence between the mtDNAs (5), whereas extracts D and E were prepared for this work from
of the Neandertal and contemporary humans is estimated to 0.4 g of bone each. Extracts A, B, and C were known to yield
465,000 years before the present, with confidence limits of PCR products that contained various proportions of modern
317,000 and 741,000 years. Taken together, the results support human mtDNA sequences in addition to the Neandertal
the concept that the Neandertal mtDNA evolved separately sequence (5). To test the degree of contamination of extracts
from that of modern humans for a substantial amount of time D and E, PCRs were performed for a part of HVRI, which had
and lends no support to the idea that they contributed mtDNA been determined previously (primers L16209 and H16271; ref.
to contemporary modern humans. 5). The PCR products were cloned, and 10 clones each were
sequenced. In both extracts, 8 of 10 clones carried 7 substitu-
tions and an adenosine insertion was determined for the
The role of Neandertals with respect to the evolution of
Neandertal in this region (5), whereas two clones were iden-
anatomically modern humans is controversial. Although some
tical to the reference sequence (data not shown). Thus, as in
paleontologists view Neandertals as a distinct branch in hom-
the case of extracts A and B, a small proportion of contami-
inid evolution that became extinct without any direct genetic
nating sequences is present in extracts D and E. Extract C,
contribution to present-day humans (1), others consider the which was prepared in another laboratory, contains a majority
Neandertals to be among the direct ancestors of modern of contaminating modern human DNA (5). Thus, whenever
Europeans (2). Recently, as a part of an interdisciplinary this extract was used, primers specific for putative Neandertal
project of the Rheinisches Landesmuseum Bonn (3, 4), the sequences determined from adjacent segments were used (cf.
DNA sequence of the first hypervariable region (HVRI) of the Fig. 1). There were 6, 2, and 19 clones derived from amplifi-
mtDNA from the Neandertal type specimen was determined cations C21, C23, and E24 (Fig. 1), respectively, that contained
(5). When compared with HVRI sequences of contemporary sequences with only one or no difference from the reference
humans, the Neandertal mtDNA tended to fall outside the sequence (7). These clones (not shown) were considered
variation of modern humans. Furthermore, phylogenetic anal- contaminants and were not included in the reconstruction of
yses suggested that the Neandertal mtDNA was an outgroup the Neandertal sequence. For the other amplifications, all
to the mtDNAs of modern humans, and the age of the most clones sequenced and the primers used are shown in Fig. 1.
recent common ancestor (MRCA) of the mtDNAs of the Alignments and Sequence Analyses. For the analysis of the
Neandertal and modern humans was estimated to be about HVRI and HVRII sequences, the Neandertal sequences were
four times older than the age of the MRCA of modern human aligned to a data set of 663 contemporary mtDNA lineages,
mtDNAs. These results indicate that the Neandertal mtDNA i.e., distinct mtDNA sequences found among 682 contempo-
gene pool evolved for a substantial time period as an entity rary humans (8). All human sequences with ambiguities in the
distinct from modern humans and give no indication that reported sequences were excluded before the analysis. In
Neandertals contributed mtDNA to modern humans (5, 6). addition, nine mtDNA lineages from seven common chimpan-
However, because these analyses were based on a DNA zees and two bonobos (‘‘pygmy chimpanzees’’) were used
sequence of only 333 bp, the results are less than conclusive. (9–14). At positions where insertions deletions occurred be-
For example, the support for the placement of the Neandertal tween the sequences of the apes, humans and the Neandertal
mtDNA outside the variation of modern human mtDNA in the were excluded from the alignment. Sequence comparisons thus
phylogenetic tree was merely 89% (5). To better estimate the were based on a total of 600 nucleotide positions, encompass-
relationship of the Neandertal mtDNA to the current mtDNA ing positions 16024–16365 and 73–340, but excluding positions
gene pool, we have determined 340 bp of the second mtDNA
HVR (HVRII) from the Neandertal type specimen and ana- This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: HVRI and HVRII, first and second hypervariable
The publication costs of this article were defrayed in part by page charge region, respectively; MRCA, most recent common ancestor.
Data deposition: The sequence reported in this paper has been
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
deposited in the GenBank database (accession no. AF142095).
accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. e-mail: paabo@eva.
PNAS is available online at www.pnas.org. mpg.de.
5581
3. 5582 Evolution: Krings et al. Proc. Natl. Acad. Sci. USA 96 (1999)
16078, 16166, 252, 291, 299, and 317–321 (numbering accord- stitutions was not found, although all four positions are
ing to ref. 7). Pairwise sequence differences were calculated by variable among humans.
using unpublished software by A. von Haeseler (Max Planck To determine the rest of the HVRII sequence, additional
Institute for Evolutionary Anthropology Leipzig, Germany). amplifications partly overlapping with the above segment were
The transition transversion ratio was estimated by using the performed from three different extracts, one of which had
663 human lineages and the program PUZZLE 4.0 (15), and been prepared in another laboratory (extract C, ref. 5). All
genetic distances between pairs of sequences were calculated sequence positions from position 57 to 396 were scored by
according to the F84 model using the ‘‘ML’’ option of the using 9–21 clones from at least two independent amplifica-
program ‘‘dnadist’’ from the PHYLIP package (16). A neighbor- tions. Nucleotide states reproducible between amplifications
joining tree (17) was produced by using the program ‘‘neigh- were inferred to exist in the original template molecules.
bor’’ from PHYLIP, and the support for branches in this tree was Authenticity. The sequence determined was considered to
calculated with the likelihood mapping option in PUZZLE 4.0 be derived from the mtDNA of the Neandertal individual for
(18). For the estimation of the age of the MRCAs, pairwise the following reasons. (i) An analysis of the state of preser-
genetic distances were calculated with the Tamura—Nei al- vation of amino acids in the bone (5) has shown that the
gorithm (19) as implemented in PUZZLE 4.0. The parameters conditions under which the bone has been preserved are
(transition transversion ratio), (purine pyrimidine transi- compatible with macromolecular preservation. (ii) The DNA
tion ratio), and , which describes the distribution of the sequence could be amplified reproducibly from different
evolutionary rates of individual nucleotide positions (20, 21), extracts. Because the amount of bone available for extractions
were estimated from the human sequences by using PUZZLE 4.0. is limited, the HVRII sequence was not reproduced from an
Means and SDs of the genetic distances within and between independent extract in another laboratory. However, this was
species were calculated by using the program EXCEL 4.0. done for the HVRI sequence determined previously (5). For
The rate of nucleotide substitution per position per year per the HVRII sequence, the extract prepared at Penn State
lineage was calculated by the method of Tamura and Nei (19), University (extract C) yielded the same sequence as extracts
using as a calibration point the divergence of humans and prepared in Munich. (iii) The DNA sequence falls as an
chimpanzees 4–5 million years ago (22, 23). The upper and outgroup to modern human sequences in phylogenetic analy-
lower confidence limits of this rate were estimated by using, ses (see below), an observation that may be taken to support
respectively, the upper 95% confidence limit of the mean that it is derived from the bone. However, in some cases,
genetic distance and the younger date for the human— divergent mtDNA sequences derived from amplifications of
chimpanzee split, and the lower confidence limit of the genetic contemporary DNA containing nuclear insertions of mtDNA
distance and the older date for the human—chimpanzee split. segments have been misidentified as ancient sequences (26).
These two rates, in turn, were used to calculate the lower and Therefore, we designed a primer pair (NL152, cf. Fig. 1 and
upper limits of the age of the MRCA of the human and NH243 5 -TGG CTG TGC A A CAT TTA GTC-3 ) that
chimpanzee mtDNA gene pools. matches the sequence from the Neandertal type specimen and
not contemporary human mtDNA sequences. Under amplifi-
cation conditions that allow less than one copy of the Nean-
RESULTS AND DISCUSSION dertal sequence per genome of human genomic DNA to be
Retrieval of the Neandertal HVRII. Primers designed to amplified, these primers failed to produce products in ampli-
amplify human and chimpanzee mtDNA sequences were used fications attempted from nine Africans, six Europeans, eight
to amplify a 122-bp segment (including primers) of HVRII Asians, and three Australians Oceanians. This makes a nu-
from extract B, prepared from the 0.4 g of the right humerus clear insertion an unlikely source of the sequence. (iv) If some
of the Neandertal type specimen (24). A weak amplification form of miscoding DNA damage that was highly sequence-
product could be visualized on an agarose gel. This product specific were prevalent in the Neandertal DNA molecules, this
was reamplified and cloned in a plasmid vector, and the inserts would result in nucleotide substitutions that would be repro-
of 13 clones were sequenced (Fig. 1, B18). All clones carried duced between independent amplifications and thus would be
four identical substitutions from the contemporary human mistaken for substitutions in the authentic Neandertal DNA
reference sequence (7). Furthermore, two other positions sequence. We consider this unlikely because 37 of 38 positions
differed from the reference sequence in six and seven clones, in which a substitutional difference between the Neandertal
respectively, and four more positions showed substitutions in and reference sequence are observed in HVRI and HVRII
single clones. The same primers were used to amplify the same also show differences among modern humans, chimpanzees,
DNA segment from a different extract (Fig. 1, D20). Among and bonobos. Because 295 of 600 positions studied are variable
the seven clones sequenced, the four substitutions found in all in this data set, this would be an extremely unlikely result (P
clones of the first amplification were found in all of these 1.13 10 11) if the substitutions in the Neandertal were
clones, with the sole exception of a G at position 189 in one of generated by a process different from the process generating
the clones. Neither the four singleton substitutions nor the two the differences in the contemporary species, for example, some
substitutions observed in several clones from the first ampli- form of postmortem chemical damage.
fication were seen among the clones from the second ampli- Sequence Comparisons. Among the 340 positions deter-
fication. However, three more singleton substitutions were mined for HVRII, 11 transitional differences from the refer-
observed at positions that showed no variation among the first ence sequence (7) were identified. In addition, an insertion of
amplification. Substitutions that are not reproducible between three thymine residues occurs in a C-rich region after position
amplifications are likely to be due to nucleotide misincorpo- 307 that shows length variation in humans (8).
rations during the PCR, which may be induced by damage To estimate the relationship of the Neandertal mtDNA to
present in ancient DNA (25). That two nonreproducible that of contemporary humans, positions 73–340 of HVRII
substitutions occur in a large proportion of clones from one of were joined with positions 16024–16365 of HVRI and aligned
the amplifications indicates that the amplifications start from to the homologous sequences from 151 Africans, 472 Euro-
very few template molecules, a supposition that agrees with the peans, 41 Asians, 10 Native Americans, and 15 Australian
quantitation performed previously (5). When the sequence Oceanians as well as 7 chimpanzees and 2 bonobos. Positions
with the four substitutions seen in both amplifications was at which insertions deletions occurred were excluded. Some
compared with a collection of 951 contemporary human humans shared the same sequences such that the data set could
mtDNA control region lineages (8), this combination of sub- be collapsed to 663 different mitochondrial lineages.
4. Evolution: Krings et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5583
Table 1. Pairwise sequence differences between the Neandertal,
humans, and some apes
Chimpanzees
Humans Neandertal bonobos
(n 663) (n 1) (n 9)
Humans 10.9 5.1 35.3 2.3 93.4 7.1
(1–35) (29–43) (78–113)
Neandertal 94.1 5.7
(84–103)
Chimpanzees 54.8 24
bonobos (1–81)
Shown are means SD and ranges (in parentheses) of pairwise
sequence differences. The chimpanzee and bonobo DNA sequences
used come from two P. troglodytes troglodytes, one P. troglodytes verus,
four P. troglodytes spec., and two bonobos (Pan paniscus).
The contemporary human mtDNA lineages differ at an
average of 10.9 positions from one another and at 35.3
positions from the Neandertal (Table 1). Thus, on average, the
Neandertal mtDNA has more than three times as many
differences from modern human sequences as the latter have
between them. In addition to the substitutions, the Neandertal
sequences carry an insertion of an A after position 16263 in
HVRI as well as the insertion of three T residues in HVRII.
It may be noted that a small fraction (0.037%) of the inter-
human comparisons are larger than the smallest distance (29
substitutions) between the Neandertal and humans.
It has been suggested that Neandertals were among the
direct ancestors of modern Europeans (2, 27). European
mtDNAs therefore might be expected to have fewer nucleotide
differences from the Neandertal mtDNA than mtDNAs from
Africa or Asia. The modern human sequences from the
different continents therefore were compared separately with
the Neandertal sequence. The mtDNAs from Africa, Europe,
and Asia were found to carry 34.4 2.7, 35.8 2.1, and 33.8
2.0 differences from the Neandertal sequence, respectively.
The modern human lineages displaying the fewest differences
(29 substitutions) to the Neandertal mtDNA were found in
Africa, but the closest lineages in Asia and Europe were almost
as similar to the Neandertal (30 and 31 differences, respec-
tively). Thus, the mtDNA gene pools of modern humans on
these continents are equidistant from the Neandertal mtDNA.
Because of the stochastic nature of the accumulation of
mutations and the heterogeneity of substitution rates among
nucleotide positions, it is not surprising that some contempo-
rary mtDNAs have fewer differences from the Neandertal
mtDNA than the maximum number of differences seen among
contemporary mtDNAs. This cannot be taken as an indication
that these contemporary mtDNAs are more closely related in
a phylogenetic sense to the Neandertal mtDNA than they are
to other contemporary mtDNAs, as has been implied recently
(28). Rather, phylogenetic analyses are needed to elucidate
this question.
Phylogenetic Analysis. The observed nucleotide differences
among all pairs of sequences of the Neandertal, the 663
modern humans, and the 7 chimpanzees and 2 bonobos were
FIG. 1. HVRII mtDNA sequences of clones derived from PCR
products from Neandertal bone extracts. Dots indicate base identity to
the human reference sequence (7). Clone designations are made up of
a letter (B, C, D, E) that refers to DNA extracts followed by the
number of the amplification performed and, after the dot, the number
of the individual clones. Primers used are given for the first clone of
each PCR. Numbers in primer designations indicate the number of the
3 base according to ref. 7, and L and H refer to the light and heavy
strands of mtDNA. The sequences of the H primers are reverse and
complemented. Standard designations are used for unidentified bases.
The numbers of the positions at which substitutions in the Neandertal
were found are indicated.
5. 5584 Evolution: Krings et al. Proc. Natl. Acad. Sci. USA 96 (1999)
corrected for multiple substitutions, and a phylogenetic tree the two bonobos was calculated as 2,844,000 years (confidence
was constructed by using the neighbor-joining algorithm (17). limits: 1,940,000 and 4,534,000 years).
In this tree the Neandertal forms the outgroup to the modern Relative Divergence Between Neandertals and Humans. In
human mtDNAs (Fig. 2). The reliability of the branch con- western Europe, Neandertals and modern humans coexisted
necting the Neandertal mtDNA with that of the modern from approximately 40,000 years ago to less than 30,000 years
humans was tested by the likelihood-mapping approach (18), ago (34). The implications of that coexistence in terms of
where quartets of sequences involving the Neandertal, a culture and genetic relationships are a matter of debate. The
chimpanzee or bonobo, and two humans were analyzed such results presented here indicate that the mtDNA gene pools of
that the probability of sampling each human sequence at least these two hominid forms had diverged for a substantial time
once was 0.96. For each quartet, the likelihood of each of the before they came into contact. To put the extent of genetic
three possible tree topologies was calculated. In 100% of the differentiation that had resulted into perspective, a useful
1,898,505 quartets tested the most likely topology had the comparison may be the differentiation found today among
Neandertal mtDNA as an outgroup to that of the humans. chimpanzees and bonobos. The number of differences between
Furthermore, among the modern human mtDNAs, nine and the Neandertal and modern humans is 35.5 2.3, about half
eight African sequences were found to branch off on the first that between chimpanzees and bonobos (75.7 4.6). Unfor-
and the second branch after the Neandertal, respectively. tunately, HVRII sequences are not available for different
These branches were supported by 91% and 92% of quartets, subspecies of chimpanzees. However, if the analysis is confined
respectively. to 312 bp of HVRI, the average difference between modern
Thus, the phylogenetic analysis shows that the line leading humans and the Neandertal is 25.6 2.2, whereas that among
to the Neandertal mtDNA diverged before the most recent 19 bonobos is 17.7 8.5, among 10 central chimpanzees (Pan
common ancestor of the modern human mtDNA gene pool troglodytes troglodytes) is 14.6 8.1, among 25 western chim-
existed. Furthermore, as has been pointed out earlier (5, panzees (P. troglodytes verus) is 21.8 9.7, and among 108
29–31), the branching pattern among human mtDNAs sug- eastern chimpanzees (P. troglodytes schweinfurthii) is 7.9 3.0.
gests an African origin of the modern human mtDNA gene The observed differences between the subspecies varies from
pool. 19.7 2.9 between central and eastern chimpanzees and
Dates of Divergences. For the estimation of the ages of 36.2 6.1 and 33.0 4.5 between western and central, and
MRCAs of different groups of mtDNAs, the observed nucle- western and eastern chimpanzees, respectively. Thus, the
otide differences were corrected for multiple substitutions by
average observed difference between the Neandertal mtDNA
using the Tamura–Nei algorithm (17). The resulting genetic
and the mtDNA of modern humans exceeds that occurring
distances and the estimated age of the modern human–
within chimpanzee subspecies and within bonobos, but is less
chimpanzee split of 4–5 million years (22, 23) were used to
than what is found between two of three pairwise comparisons
calculate the substitution rate of 0.94 10 7 substitutions per
between currently recognized subspecies of chimpanzees.
site per year per lineage with 5.92 10 8 and 1.38 10 7 as
When the differences are corrected for multiple substitutions,
the lower and upper confidence limits. These estimates are in
reasonable agreement with previous rate estimations for the this general situation remains unchanged. However, mtDNA
mtDNA control region (32, 33). Using these rates, the age of sequences from more Neandertal individuals are needed to
the MRCA of the Neandertal and modern human mtDNAs obtain a better understanding of the extent of separation
was estimated to be 465,000 years, with confidence limits of between the mtDNA gene pools of Neandertals and modern
317,000 and 741,000 years. This age is significantly older than humans.
that of the MRCA of modern human mtDNAs, which, by the
same procedure, was determined to be 163,000 years, with CONCLUSIONS
111,000 and 260,000 years as confidence limits. Finally, the age
of the MRCA of the mtDNAs of the seven chimpanzees and The divergence of the Neandertal mtDNA from the line
leading to the contemporary human mtDNA gene pool is
almost 3-fold older than the deepest divergence among con-
temporary human mtDNAs. The extent of sequence diver-
gence exceeds that found within current chimpanzee subspe-
cies. This shows that the Neandertal mtDNA and the human
ancestral mtDNA gene pool have evolved as separate entities
for a substantial period of time and gives no support to the
notion that Neandertals should have contributed mtDNA to
the modern human gene pool.
We are indebted to F. G. Zehnder and H.-E. Joachim (Rheinisches
Landesmuseum Bonn) for permission to remove samples, to I. Boschi
and V. Pascali for unpublished human mtDNA sequences, to P.
Gagneux and U. Gerloff for unpublished chimpanzee and bonobo
mtDNA sequences, to C. Capelli, S. Meyer, K. Strimmer, L. Vigilant,
A. von Haeseler, and W. Schartau for discussion and technical help,
and to the Boehringer–Ingelheim Fonds and the Deutsche For-
schungsgemeinschaft for financial support.
1. Stringer, C. & McKie, R. (1996) African Exodus (Random House,
London).
2. Wolpoff, M. & Caspari, R. (1997) Race and Human Evolution
(Simon & Schuster, New York).
FIG. 2. A schematic phylogenetic tree of the Neandertal mtDNA 3. Schmitz, R. W., Pieper, P., Bonte, W. & Krainitzki, H. (1995) in
and 663 modern human mtDNA lineages. Only the relevant branches Advances in Forensic Sciences, Forensic Odontology, eds. Jacob, B.
are shown. The tree was rooted with seven chimpanzees and two & Bonte, W. (Koster, Berlin), Vol. 7, pp. 42–44.
¨
bonobos. Numbers on internal branches indicate quartet puzzling 4. Schmitz, R. W. (1996) Doctoral thesis (University of Cologne,
support values (18). Germany).
6. Evolution: Krings et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5585
5. Krings, M., Stone, A., Schmitz, R.-W., Krainitzki, H., Stoneking, 19. Tamura, K. & Nei, M. (1993) J. Mol. Evol. 10, 512–526.
M. & Pa¨bo, S. (1997) Cell 90, 19–30.
¨a 20. Wakeley, J. (1993) J. Mol. Evol. 37, 613–623.
6. Nordborg, M. (1998) Am. J. Hum. Genet. 63, 1237–1240. 21. Yang, Z. (1994) J. Mol. Evol. 39, 306–314.
7. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., 22. Adachi, J. & Hasegawa, M. (1995) J. Mol. Evol. 40, 622–628.
Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, 23. Takahata, N., Satta, Y. & Klein, J. (1995) Theor. Popul. Biol. 48,
B. A., Sanger, F., et al. (1981) Nature (London) 290, 457–474. 198–221.
8. Handt, O., Meyer, S. & von Haeseler, A. (1998) Nucleic Acids Res. 24. King, W. (1864) Q. J. Sci. 1, 88–97.
26, 126–129. 25. Hoss, M., Jaruga, P., Zastawny, T. H., Dizdaroglu, M. & Pa¨bo,
¨ ¨a
9. Arnason, U., Xu, X. & Gullberg, A. (1996) J. Mol. Evol. 42, S. (1996) Nucleic Acids Res. 24, 1304–1307.
145–152. 26. Zischler, H., Hoss, M., Handt, O., von Haeseler, A., van der Kuyl,
¨
10. Foran, D. R., Hixson, J. E. & Brown, W. M. (1988) Nucleic Acids A. C., Goudsmit, J. & Pa¨bo, S. (1995) Science 268, 1193.
¨a
Res. 16, 5841–5861. 27. Smith, F. H., Falsetti, A. B. & Donelly, S. M. (1989) Yearbook
11. Goldberg, T. L. & Ruvolo, M. (1997) Mol. Biol. Evol. 14, 976–984. Phys. Anthropol. 32, 35–68.
12. Horai, S., Hayasaka, K., Kondo, R., Tsugane, K. & Takahata, N. 28. Wolpoff, M. (1998) Evol. Anthropol. 7, 1–3.
(1995) Proc. Natl. Acad. Sci. USA 92, 532–536. 29. Cann, R. L., Stoneking, M. & Wilson, A. C. (1987) Nature
13. Kocher, T. D. & Wilson, A. C. (1991) in Evolution of Life: Fossils, (London) 325, 31–36.
Molecules and Culture, eds. Osawa, S. & Honjo, T. (Springer, 30. Vigilant, L., Stoneking, M., Harpending, H., Hawkes, K. &
Tokyo), pp. 391–413. Wilson, A. C. (1991) Science 253, 1503–1507.
14. Morin, P. A., Moore, J. J., Chakraborty, R., Jin, L., Goodall, J. 31. Zischler, H., Geisert, H., von Haeseler, A. & Pa¨bo, S. (1995)
¨a
& Woodruff, D. S. (1994) Science 265, 1193–1201. Nature (London) 378, 489–492.
15. Strimmer, K. & von Haeseler, A. (1996) J. Mol. Evol. 13, 964–969. 32. Jazin, E., Soodyall, H., Jalonen, P., Lindholm, E., Stoneking, M.
16. Felsenstein, J. (1994) PHYLIP (University of Washington, Seattle), & Gyllensten, U. (1998) Nat. Genet. 18, 109–110.
Version 3.5. 33. Ward, R. H., Frazier, B. L., Dew-Jager, K. & Pa¨bo, S. (1991)
¨a
17. Saitou, N. & Nei, M. (1987) Mol. Biol. Evol. 4, 406–425. Proc. Natl. Acad. Sci. USA 88, 8720–8724.
18. Strimmer, K. & von Haeseler, A. (1997) Proc. Natl. Acad. Sci. 34. Hublin, J.-J., Barosso Ruiz, C., Medina Lara, P., Fontugne, M. &
USA 94, 6815–6819. Reyss, J.-L. (1995) C. R. Acad. Sci. Paris 321, 931–937.