Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development ... More
Cell contact-dependent priming and Fc interaction with CD32+ immune cells contribute to the TGN1412-triggered cytokine response.
'Following inconspicuous preclinical testing, the superagonistic anti-CD28 mAb TGN1412 was applied to six study participants who all developed a devastating cytokine storm. We verified that TGN1412 treatment of fresh PBMCs induced only moderate responses, whereas restoration of tissue-like conditions by high-density preculture (HDC) allowed vigorous cytokine production. TGN1412 treatment of ... More
High transcript level of fatty acid-binding protein 11 but not of very low-density lipoprotein receptor is correlated to ovarian follicle atresia in a teleost fish (Solea senegalensis).
AuthorsAgulleiro MJ, André M, Morais S, Cerdà J, Babin PJ,
JournalBiol Reprod
PubMed ID17554079
'Transcripts encoding a fatty acid-binding protein (FABP), Fabp11, and two isoforms of very low-density lipoprotein receptor (Vldlr; vitellogenin receptor) were characterized from the ovary of Senegalese sole (Solea senegalensis). Phylogenetic analyses of vertebrate FABPs demonstrated that Senegalese sole Fabp11, as zebrafish (Danio rerio) homologous sequences, is part of a newly ... More
[DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors].
'The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San ... More
Prevalence of Toxoplasma gondii infection in stray and household cats in regions of Seoul, Korea.
AuthorsLee SE, Kim JY, Kim YA, Cho SH, Ahn HJ, Woo HM, Lee WJ, Nam HW,
JournalKorean J Parasitol
PubMed ID20877509
'The principal objective of this study was to investigate the prevalence of toxoplasmosis in household and stray cats in Seoul, Republic of Korea. We collected blood samples from 72 stray and 80 household cats, and all samples were examined by ELISA and nested PCR. The overall positive rates of Toxoplasma ... More
Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing.
AuthorsSharp CE, Stott MB, Dunfield PF,
JournalFront Microbiol
PubMed ID22912630
Genomic analysis of the methanotrophic verrucomicrobium
Draft Genome Sequences of Six Rhodobacter capsulatus Strains, YW1, YW2, B6, Y262, R121, and DE442.
AuthorsDing H, Moksa MM, Hirst M, Beatty JT,
Journal
PubMed ID24526637
Rhodobacter capsulatus is a model organism for studying a novel type of horizontal gene transfer mediated by a phage-like gene transfer agent (RcGTA). Here we report the draft genome sequences of six R. capsulatus strains that exhibit different RcGTA properties, including RcGTA overproducers, RcGTA nonproducers, and/or RcGTA nonreceivers. ... More
Mutations in EZH2 cause Weaver syndrome.
AuthorsGibson WT, Hood RL, Zhan SH, Bulman DE, Fejes AP, Moore R, Mungall AJ, Eydoux P, Babul-Hirji R, An J, Marra MA, Chitayat D, Boycott KM, Weaver DD, Jones SJ,
JournalAm J Hum Genet
PubMed ID22177091
We used trio-based whole-exome sequencing to analyze two families affected by Weaver syndrome, including one of the original families reported in 1974. Filtering of rare variants in the affected probands against the parental variants identified two different de novo mutations in the enhancer of zeste homolog 2 (EZH2). Sanger sequencing ... More
Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.
AuthorsArakaki A, Shibusawa M, Hosokawa M, Matsunaga T,
JournalAppl Environ Microbiol
PubMed ID20081000
Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order ... More
Multigene profiling to identify alternative treatment options for glioblastoma: a pilot study.
: Glioblastoma (GBM) is a highly aggressive malignancy and the most effective treatment regime has a high relapse rate. Increasingly, the development of therapies involves defining drug-diagnostic combinations where the presence of a molecular target or marker identifies patients who are most likely to respond to a specific therapy. Trials ... More
Network quantification of EGFR signaling unveils potential for targeted combination therapy.
AuthorsKlinger B, Sieber A, Fritsche-Guenther R, Witzel F, Berry L, Schumacher D, Yan Y, Durek P, Merchant M, Schäfer R, Sers C, Blüthgen N,
Journal
PubMed ID23752269
The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, ... More
Automated extraction improves multiplex molecular detection of infection in septic patients.
AuthorsRegueiro BJ, Varela-Ledo E, Martinez-Lamas L, Rodriguez-Calviño J, Aguilera A, Santos A, Gomez-Tato A, Alvarez-Escudero J,
JournalPLoS One
PubMed ID20967222
Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test M(grade) (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 ... More
Development and validation of a SYBR-Green I real-time PCR protocol to detect hazelnut (Corylus avellana L.) in foods through calibration via plasmid reference standard.
AuthorsD'Andrea M, Coïsson JD, Travaglia F, Garino C, Arlorio M,
JournalJ Agric Food Chem
PubMed ID19891480
Many tree nuts are considered to be a serious problem in food safety, because of the presence of causative factors in IgE-mediated food allergies. Among these, hazelnut (Corylus avellana L.) seeds are largely used in a range of confectionery products and contain many well-characterized allergens. DNA-based methods and ELISA tests ... More
Exposure of DNA and Bacillus subtilis spores to simulated martian environments: use of quantitative PCR (qPCR) to measure inactivation rates of DNA to function as a template molecule.
Several NASA and ESA missions are planned for the next decade to investigate the possibility of present or past life on Mars. Evidence of extraterrestrial life will likely rely on the detection of biomolecules, which highlights the importance of preventing forward contamination not only with viable microorganisms but also with ... More
Sensitive multiplex real-time reverse transcription-PCR assay for the detection of human and animal noroviruses in clinical and environmental samples.
AuthorsWolf S, Williamson WM, Hewitt J, Rivera-Aban M, Lin S, Ball A, Scholes P, Greening GE,
JournalAppl Environ Microbiol
PubMed ID17616614
In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishes between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three ... More
DNA qualification workflow for next generation sequencing of histopathological samples.
AuthorsSimbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpeli G, Lawlor RT, Scarpa A,
Journal
PubMed ID23762227
Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the ... More
The DNA methylation landscape of human early embryos.
AuthorsGuo H, Zhu P, Yan L, Li R, Hu B, Lian Y, Yan J, Ren X, Lin S, Li J, Jin X, Shi X, Liu P, Wang X, Wang W, Wei Y, Li X, Guo F, Wu X, Fan X, Yong J, Wen L, Xie SX, Tang F, Qiao J
Journal
PubMed ID25079557
DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos ... More
A comparative analysis of drinking water employing metagenomics.
AuthorsBrumfield KD, Hasan NA, Leddy MB, Cotruvo JA, Rashed SM, Colwell RR, Huq A
JournalPLoS One
PubMed ID32271799
'The microbiological content of drinking water traditionally is determined by employing culture-dependent methods that are unable to detect all microorganisms, especially those that are not culturable. High-throughput sequencing now makes it possible to determine the microbiome of drinking water. Thus, the natural microbiota of water and water distribution systems can ... More
Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription.
AuthorsXiao R, Chen JY, Liang Z, Luo D, Chen G, Lu ZJ, Chen Y, Zhou B, Li H, Du X, Yang Y, San M, Wei X, Liu W, Lécuyer E, Graveley BR, Yeo GW, Burge CB, Zhang MQ, Zhou Y, Fu XD
JournalCell
PubMed ID31251911
'Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing ... More
Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples.
AuthorsHe HJ, Stein EV, DeRose P, Cole KD
JournalBiotechniques
PubMed ID29571283
'We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers ... More
DNA isolation from achieved formalin-fixed paraffin-embedded tissues in a series of 212 thyroid carcinoma cases: the influence of preanalytical factors on DNA quantity and purity.
AuthorsNechifor-Boila A, Banescu C, Zahan AE, Moldovan V, Szasz E, Borda A
JournalJ Investig Med
PubMed ID31801793
'The present study aimed to investigate the influence of several important preanalytical factors (storage period of the tumor block, maximal diameter of the tumor circled area, tumor volume and tumor fraction) on the isolated DNA from formalin-fixed paraffin-embedded (FFPE) tissues in a series of thyroid carcinomas.' ... More
RNA sequencing by direct tagmentation of RNA/DNA hybrids.
AuthorsDi L, Fu Y, Sun Y, Li J, Liu L, Yao J, Wang G, Wu Y, Lao K, Lee RW, Zheng G, Xu J, Oh J, Wang D, Xie XS, Huang Y, Wang J
JournalProc Natl Acad Sci U S A
PubMed ID31988135
'Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries ... More
Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.
AuthorsRanjan R, Rani A, Metwally A, McGee HS, Perkins DL
JournalBiochem Biophys Res Commun
PubMed ID26718401
'The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have ... More
Design of a Targeted Sequencing Assay to Detect Rare Mutations in Circulating Tumor DNA.
AuthorsChen J, Chen J, He F, Huang Y, Lu S, Fan H, Wang M, Xu R
JournalGenet Test Mol Biomarkers
PubMed ID30986100
'Qualitative and quantitative detection of circulating tumor DNA (ctDNA) is a liquid biopsy technology used for early cancer diagnosis. However, the plasma ctDNA content is extremely low, so it is difficult to detect somatic mutations of tumors using conventional sequencing methods. Target region sequencing (TRS) technology, through enrichment of the ... More
Quantification of DNA Extracted from Formalin Fixed Paraffin-Embeded Tissue Comparison of Three Techniques: Effect on PCR Efficiency.
AuthorsKumar D, Panigrahi MK, Suryavanshi M, Mehta A, Saikia KK
JournalJ Clin Diagn Res
PubMed ID27790419
Mutation detection from Formalin Fixed Paraffin-Embedding (FFPE) tissue in molecular lab became a necessary tool for defining potential targeted drug. Accurate quantification of DNA extracted from FFPE tissue is necessary for downstream applications like Polymerase Chain Reaction (PCR), sequencing etc. ... More
Sensitive tumour detection and classification using plasma cell-free DNA methylomes.
AuthorsShen SY, Singhania R, Fehringer G, Chakravarthy A, Roehrl MHA, Chadwick D, Zuzarte PC, Borgida A, Wang TT, Li T, Kis O, Zhao Z, Spreafico A, Medina TDS, Wang Y, Roulois D, Ettayebi I, Chen Z, Chow S, Murphy T, Arruda A, O'Kane GM, Liu J, Mansour M, McPherson JD, O'Brien C, Leighl N, Bedard PL, Fleshner N, Liu G, Minden MD, Gallinger S, Goldenberg A, Pugh TJ, Hoffman MM, Bratman SV, Hung RJ, De Carvalho DD
JournalNature
PubMed ID30429608
The use of liquid biopsies for cancer detection and management is rapidly gaining prominence
CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway.
AuthorsRichardson CD, Kazane KR, Feng SJ, Zelin E, Bray NL, Schäfer AJ, Floor SN, Corn JE
JournalNat Genet
PubMed ID30054595
CRISPR-Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in ... More
AuthorsChen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA
JournalScience
PubMed ID29449511
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA ... More
Whole genome sequencing analysis of multiple Salmonella serovars provides insights into phylogenetic relatedness, antimicrobial resistance, and virulence markers across humans, food animals and agriculture environmental sources.
AuthorsPornsukarom S, van Vliet AHM, Thakur S,
JournalBMC Genomics
PubMed ID30400810
Salmonella enterica is a significant foodborne pathogen, which can be transmitted via several distinct routes, and reports on acquisition of antimicrobial resistance (AMR) are increasing. To better understand the association between human Salmonella clinical isolates and the potential environmental/animal reservoirs, whole genome sequencing (WGS) was used to investigate the epidemiology ... More
Nested PCR and the TaqMan SNP Genotyping Assay enhanced the sensitivity of drug resistance testing of Mycobacterium leprae using clinical specimens of leprosy patients.
AuthorsChen X, He J, Liu J, You Y, Yuan L, Wen Y
JournalPLoS Negl Trop Dis
PubMed ID31881061
Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy ... More
Identification and characterization of a core set of ROS wave-associated transcripts involved in the systemic acquired acclimation response of Arabidopsis to excess light.
AuthorsZandalinas SI, Sengupta S, Burks D, Azad RK, Mittler R
JournalPlant J
PubMed ID30556340
Systemic acquired acclimation (SAA) plays a key role in optimizing growth and preventing damage associated with fluctuating or abrupt changes in the plant environment. To be effective, SAA has to occur at a rapid rate and depend on rapid signaling pathways that transmit signals from affected tissues to all parts ... More
Bacterial Single Cell Whole Transcriptome Amplification in Microfluidic Platform Shows Putative Gene Expression Heterogeneity.
AuthorsLiu Y, Jeraldo P, Jang JS, Eckloff B, Jen J, Walther-Antonio M
JournalAnal Chem
PubMed ID31188565
Single cell RNA sequencing is a technology that provides the capability of analyzing the transcriptome of a single cell from a population. So far, single cell RNA sequencing has been focused mostly on human cells due to the larger starting amount of RNA template for subsequent amplification. One of the ... More
A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.
AuthorsMaji B, Gangopadhyay SA, Lee M, Shi M, Wu P, Heler R, Mok B, Lim D, Siriwardena SU, Paul B, Dancík V, Vetere A, Mesleh MF, Marraffini LA, Liu DR, Clemons PA, Wagner BK, Choudhary A
JournalCell
PubMed ID31051099
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite ... More
A step-by-step beginner's protocol for whole genome sequencing of human bacterial pathogens.
AuthorsGautam SS, Kc R, Leong KW, Mac Aogáin M, O'Toole RF
JournalJ Biol Methods
PubMed ID31453259
Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms ... More
SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.
AuthorsZiegler CGK, Allon SJ, Nyquist SK, Mbano IM, Miao VN, Tzouanas CN, Cao Y, Yousif AS, Bals J, Hauser BM, Feldman J, Muus C, Wadsworth MH, Kazer SW, Hughes TK, Doran B, Gatter GJ, Vukovic M, Taliaferro F, Mead BE, Guo Z, Wang JP, Gras D, Plaisant M, Ansari M, Angelidis I, Adler H, Sucre JMS, Taylor CJ, Lin B, Waghray A, Mitsialis V, Dwyer DF, Buchheit KM, Boyce JA, Barrett NA, Laidlaw TM, Carroll SL, Colonna L, Tkachev V, Peterson CW, Yu A, Zheng HB, Gideon HP, Winchell CG, Lin PL, Bingle CD, Snap
JournalCell
PubMed ID32413319
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets ... More
Intratumoral heterogeneity and clonal evolution in liver cancer.
AuthorsLosic B, Craig AJ, Villacorta-Martin C, Martins-Filho SN, Akers N, Chen X, Ahsen ME, von Felden J, Labgaa I, D'Avola D, Allette K, Lira SA, Furtado GC, Garcia-Lezana T, Restrepo P, Stueck A, Ward SC, Fiel MI, Hiotis SP, Gunasekaran G, Sia D, Schadt EE, Sebra R, Schwartz M, Llovet JM, Thung S, Stolovitzky G, Villanueva A
JournalNat Commun
PubMed ID31941899
Clonal evolution of a tumor ecosystem depends on different selection pressures that are principally immune and treatment mediated. We integrate RNA-seq, DNA sequencing, TCR-seq and SNP array data across multiple regions of liver cancer specimens to map spatio-temporal interactions between cancer and immune cells. We investigate how these interactions reflect ... More
Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.
AuthorsChan-Seng-Yue M, Kim JC, Wilson GW, Ng K, Figueroa EF, O'Kane GM, Connor AA, Denroche RE, Grant RC, McLeod J, Wilson JM, Jang GH, Zhang A, Dodd A, Liang SB, Borgida A, Chadwick D, Kalimuthu S, Lungu I, Bartlett JMS, Krzyzanowski PM, Sandhu V, Tiriac H, Froeling FEM, Karasinska JM, Topham JT, Renouf DJ, Schaeffer DF, Jones SJM, Marra MA, Laskin J, Chetty R, Stein LD, Zogopoulos G, Haibe-Kains B, Campbell PJ, Tuveson DA, Knox JJ, Fischer SE, Gallinger S, Notta F
JournalNat Genet
PubMed ID31932696
Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary ... More
Highly multiplexed single-cell RNA-seq by DNA oligonucleotide tagging of cellular proteins.
AuthorsGehring J, Hwee Park J, Chen S, Thomson M, Pachter L
JournalNat Biotechnol
PubMed ID31873215
We describe a universal sample multiplexing method for single-cell RNA sequencing in which fixed cells are chemically labeled by attaching identifying DNA oligonucleotides to cellular proteins. Analysis of a 96-plex perturbation experiment revealed changes in cell population structure and transcriptional states that cannot be discerned from bulk measurements, establishing an ... More
Transmission of antimicrobial resistant non-O157 Escherichia coli at the interface of animal-fresh produce in sustainable farming environments.
AuthorsGlaize A, Gutierrez-Rodriguez E, Hanning I, Díaz-Sánchez S, Gunter C, van Vliet AHM, Watson W, Thakur S
JournalInt J Food Microbiol
PubMed ID31901751
The interaction of typical host adapted enteric bacterial pathogens with fresh produce grown in fields is complex. These interactions can be more pronounced in co-managed or sustainable farms where animal operations are, by design, close to fresh produce, and growers frequently move between the two production environments. The primary objectives ... More
Comparison of methods for the isolation of cell-free DNA from cell culture supernatant.
AuthorsBronkhorst AJ, Ungerer V, Holdenrieder S
JournalTumour Biol
PubMed ID32338581
In vitro characterization of cell-free DNA using two-dimensional cell culture models is emerging as an important step toward an improved understanding of the physical and biological characteristics of cell-free DNA in human biology. However, precise measurement of the cell-free DNA in cell culture medium is highly dependent on the efficacy ... More
Transcription polymerase-catalyzed emergence of novel RNA replicons.
AuthorsJain N, Blauch LR, Szymanski MR, Das R, Tang SKY, Yin YW, Fire AZ
JournalScience
PubMed ID32217750
Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro ... More