From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing

Meyer, Matthias; Briggs, Adrian W.; Maričić, Tomislav; Höber, Barbara; Höffner, Barbara; Krause, Johannes; Weihmann, Antje; Pääbo, Svante; Hofreiter, Michael

doi:10.1093/nar/gkm1095

Cited by 108 publications

(91 citation statements)

References 18 publications

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“…Library preparation was performed using the Roche manufacturer's protocol, eliminating the fractionation step (454; Roche). Libraries were quantitated in 20 μL qPCR reactions using methods described elsewhere (36), and amplified products were used as templates for downstream multiplex applications. Standard and multiplex PCR reactions.…”

Section: Methodsmentioning

confidence: 99%

Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid ofYersinia pestisfrom victims of the Black Death

Schuenemann

Bos

DeWitte

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Although investigations of medieval plague victims have identified Yersinia pestis as the putative etiologic agent of the pandemic, methodological limitations have prevented large-scale genomic investigations to evaluate changes in the pathogen's virulence over time. We screened over 100 skeletal remains from Black Death victims of the East Smithfield mass burial site (1348-1350, London, England). Recent methods of DNA enrichment coupled with high-throughput DNA sequencing subsequently permitted reconstruction of ten full human mitochondrial genomes (16 kb each) and the full pPCP1 (9.6 kb) virulence-associated plasmid at high coverage. Comparisons of molecular damage profiles between endogenous human and Y. pestis DNA confirmed its authenticity as an ancient pathogen, thus representing the longest contiguous genomic sequence for an ancient pathogen to date. Comparison of our reconstructed plasmid against modern Y. pestis shows identity with several isolates matching the Medievalis biovar; however, our chromosomal sequences indicate the victims were infected with a Y. pestis variant that has not been previously reported. Our data reveal that the Black Death in medieval Europe was caused by a variant of Y. pestis that may no longer exist, and genetic data carried on its pPCP1 plasmid were not responsible for the purported epidemiological differences between ancient and modern forms of Y. pestis infections.ancient DNA | paleopathology

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Section: Methodsmentioning

confidence: 99%

Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid ofYersinia pestisfrom victims of the Black Death

Schuenemann

Bos

DeWitte

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

244

209

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“…After small-fragment removal, the SrfI-digested fragment pool (in 20 l elution buffer) was used to prepare the 454 sequencing library, employing the GS Titanium library preparation kit (Roche Diagnostics). Single-stranded 454 sequencing libraries were quantified by a quantitative PCR (qPCR) assay (31) and processed by emulsion PCR, following the manufacturer's instructions. Sequencing was done on two lanes of a 70 by 75 GS Titanium picotiter plate of a Roche GS FLX sequencer.…”

Section: Methodsmentioning

confidence: 99%

Sequencing of 21 Varicella-Zoster Virus Genomes Reveals Two Novel Genotypes and Evidence of Recombination

Zell

Taudien

Pfaff

et al. 2012

J Virol

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Genotyping of 21 varicella-zoster virus (VZV) strains using a scattered single nucleotide polymorphism (SNP) method revealed ambiguous SNPs and two nontypeable isolates. For a further genetic characterization, the genomes of all strains were sequenced using the 454 technology. Almost-complete genome sequences were assembled, and most remaining gaps were closed with V aricella-zoster virus (VZV) is a member of the genus Varicellovirus within the subfamily Alphaherpesvirinae, family Herpesviridae (8). The alphaherpesviruses are characterized by their short reproduction cycle, fast spreading, efficient destruction of infected cells, and persistence in sensory ganglia. VZV replication is limited exclusively to cells of human and simian origins. The VZV genome consists of double-stranded DNA with a size of 125 kb and comprises 73 genes, 70 of which are unique and 3 of which are duplicated (9). The virus genome includes two main coding regions, unique long (U L ) and unique short (U S ), and flanking inverted repeats, termed terminal and internal repeats long (TR L , IR L ) and short (TR S , IR S ). In addition to these repeats, there are five genomic regions with tandem reiterations, designated R1 to R5.Primary infection with VZV causes varicella (chickenpox), a condition characterized by fever and exanthema with small blisters. Virus uptake occurs via the mucous membranes of the respiratory tract, with primary replication seen in the regional lymph nodes. During a short phase of primary cell-associated viremia, the virus infects peripheral blood mononuclear cells. In a secondary viremia, the virus spreads to cutaneous epithelial cells, where it induces rash. Usually, the virus is spread by excretion of aerosolized virus particles from the respiratory tract and highly infectious vesicle fluid from rash (2). After primary infection, VZV establishes a lifelong latency in trigeminal and dorsal root ganglia. Endogenous viral reactivation, e.g., after decline of VZV-specific cell-mediated immunity, may lead to viral replication and inflammation in the ganglion. Then, progeny virus migrates along the axons of neurons and is released in the skin, where it causes herpes zoster (shingles). Zoster is characterized by a unilateral vesicular rash within a single cutaneous dermatome. In most cases, the thoracic region is involved.Genetic characterization of VZV DNA is achieved by analysis of restriction fragment length polymorphism (RFLP), single nucleotide polymorphisms (SNPs), and full-genome sequencing. Early RFLP analyses revealed both interstrain variations among wild-type isolates and differences between wild-and vaccine-type viruses. The most commonly used RFLP markers of VZV included the polymorphism of open reading frames (ORFs) 38 (PstI), 54 (BglI), and 62 (SmaI) (19,23,45). These markers allowed the majority of wild-type strains in North America and Europe to be typed as PstI ϩ BglI Ϫ , whereas African and Asian strains were BglI ϩ and Japanese Oka-like wild-type strains were PstI ϩ /PstI Ϫ BglI ϩ SmaI Ϫ ; the Oka vacc...

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“…The sample-specific fragment pools were barcoded for 454 high throughput sequencing using the methods described in 43,44 . After qPCR based quantification 45 , up to six barcoded sample-specific fragment pools (6×88 fragments) were sequenced on 1/16 of a 454 GS FLX run.…”

Section: Primer Designmentioning

confidence: 99%

Discovery of lost diversity of paternal horse lineages using ancient DNA

et al. 2011

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modern domestic horses display abundant genetic diversity within female-inherited mitochondrial DnA, but practically no sequence diversity on the male-inherited Y chromosome. several hypotheses have been proposed to explain this discrepancy, but can only be tested through knowledge of the diversity in both the ancestral (pre-domestication) maternal and paternal lineages. As wild horses are practically extinct, ancient DnA studies offer the only means to assess this ancestral diversity. Here we show considerable ancestral diversity in ancient male horses by sequencing 4 kb of Y chromosomal DnA from eight ancient wild horses and one 2,800-year-old domesticated horse. Both ancient and modern domestic horses form a separate branch from the ancient wild horses, with the Przewalski horse at its base. our methodology establishes the feasibility of re-sequencing long ancient nuclear DnA fragments and demonstrates the power of ancient Y chromosome DnA sequence data to provide insights into the evolutionary history of populations.

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From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing

Cited by 108 publications

References 18 publications

Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid ofYersinia pestisfrom victims of the Black Death

Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid ofYersinia pestisfrom victims of the Black Death

Sequencing of 21 Varicella-Zoster Virus Genomes Reveals Two Novel Genotypes and Evidence of Recombination

Discovery of lost diversity of paternal horse lineages using ancient DNA

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