The Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS.
August 15, 2014 | This Thursday, a team of bioinformaticians from the National Biodefense Analysis and Countermeasures Center, the University of Maryland College Park, and sequencing company Pacific Biosciences posted information on their tool MHAP to the life sciences preprint server bioRxiv. MHAP, or MinHash Alignment Process, is a dramatically faster method for ordering DNA fragments sequenced on long-read technologies like the PacBio RS II Sequencer or the Oxford Nanopore MinION, making it easier to assemble whole genomes from scratch without the use of a reference genome.Bio-IT World previously covered MHAP following a presentation by senior author Adam Phillippy at the PacBio User Group Meeting this June; however, the newly released paper features much greater detail, including assemblies of the human genome and four important model organisms.
The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ~1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ~119 000 nearly full-length sequences and 28 000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.
Whole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material. A method is described that combines single plaque sequencing with an optimization of Sequence Independent Single Primer Amplification (SISPA). This method can be used for de novo whole genome next-generation sequencing of any cultivable virus without the need for large-scale production of viral stocks or viral purification using centrifugal techniques.
Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target Plasmodium falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by the covalent fusion of azidothymidine (AZT) with dihydroartemisinin (DHA), a tetraoxane or a 4-aminoquinoline derivative; and the small library was tested for antiviral and antimalarial activity. Our data suggests that compound 7 is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI >3000), a moderate activity against HIV (IC50 = 2.9 μM; SI >35) and not toxic to HeLa cells at concentrations used in the assay (CC50 >100 μM). Pharmacokinetics studies further revealed that compound 7 is metabolically unstable and is cleaved via O-dealkylation. These studies account for the lack of in vivo efficacy of compound 7 against the CQ-sensitive Plasmodium berghei N strain in mice, when administered orally at 20 mg/kg.
"Microbes dominate most global biogeochemical cycles, and microbial metagenomics (studying the collective microbial genomes) provides invaluable new insights into microbial systems, independent of cultivation. Metagenomic approaches targeting specific genes, e.g. small subunit (ssu) ribosomal RNA (rRNA), can be used to investigate microbial community organization by efficiently showing which taxa of organisms are present, while shotgun approaches show all genes and can indicate what functions the organisms are capable of. But collecting and organizing comprehensive shotgun data is extremely challenging and costly, and, in theory, predicting functionalities from microbial identities alone would save immense effort. However, we don’t yet know to what extent such predictions are applicable."
Finally: a population-scale means of studying microbial communities. The next few years are going to be VERY illuminating.
The results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case–control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.
The ease of creating and distributing video tutorials instead of static content improves accessibility for researchers, annotators and curators. This article focuses on online video repositories for educational and tutorial videos provided by resource developers and users. It also describes a project in Japan named TogoTV (http://togotv.dbcls.jp/en/)
PRDM9 has evolved exceptionally rapidly between humans and chimpanzees and this rapid evolution explains a second, previously published, finding of the researchers: recombination hotspots also differ between humans and chimpanzees. Remarkably, PRDM9 is involved not just in determining mouse hotspot locations, but is also a key player in mouse speciation.
Background The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome.
Findings In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs.
Conclusion Our results suggest the use of ribo-depleted RNA in a de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection.
Ed Rybicki's insight:
...and incidentally from my good friends at Stellenbosch University!
Protein microarray technology is one of the most powerful tools presently available for proteomic studies. Numerous types of protein microarrays have been widely and successfully applied for both basic biological studies and clinical researches, including those designed to characterize protein-protein, protein-nucleic acid, protein-drug/small molecule and antibody-antigen interactions. In the past decade, a variety of protein microarrays have been developed, including those spotted with whole proteomes, smaller peptides, antibodies, and lectins. Featured as high-throughput, miniaturized, and capable of parallel analysis, the power of protein microarrays has already been demonstrated many times in both basic research and clinical applications. In this review, we have summarized the latest developments in the production and application of protein microarrays. We discuss several of the most important applications of protein microarray, ranging from proteome microarrays for large scale identification of protein-protein interactions to lectin microarrays for live cell surface glycan profiling, with special emphasis on their use in studies of drug mechanisms and biomarker discovery. Already with tremendous success, we envision protein microarrays will become an indispensible tool for any systems-wide studies, fostering the integration of basic research observations to clinically useful applications.
Due to the high prevalence of viral infections having no specific treatment and the constant appearance of resistant viral strains, the development of novel antiviral agents is essential. The aim of this study was to evaluate the antiviral activity against bovine viral diarrhea virus, herpes simplex virus type 1 (HSV-1), poliovirus type 2 (PV-2) and vesicular stomatitis virus of organic (OE) and aqueous extracts (AE) from: Baccharis gaudichaudiana, B. spicata, Bidens subalternans, Pluchea sagittalis, Tagetes minuta and Tessaria absinthioides. A characterization of the antiviral activity of B. gaudichaudiana OE and AE and the bioassay-guided fractionation of the former and isolation of one active compound is also reported.
Ed Rybicki's insight:
Given my background as a plant virologist, this sort of paper always fascinates me - because I'll bet you those plants get infected with viruses.... And, of course, we have hundreds of Asteraceae around us here in SA.
Illumina shares are up today on a report that Roche is renewing its bid for the DNA sequencing leader with an offer price that has been sweetened to $8.1 billion from an earlier $6.7 billion bid. Cue Bloomberg: Illumina Inc.
We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute's (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates.
Background Human papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.
Methods Total DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.
Results Illumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence's of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6 %, 12.8 %, 4.6 % and 8.3 % respectively.
Conclusions Our results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.
Whole genome sequencing (WGS) promises to be transformative for the practice of clinical microbiology, and the rapidly falling cost and turnaround time mean that this will become a viable technology in diagnostic and reference laboratories in the near future. The objective of this article is to consider at a very practical level where, in the context of a modern diagnostic microbiology laboratory, WGS might be cost-effective compared to current alternatives. We propose that molecular epidemiology performed for surveillance and outbreak investigation and genotypic antimicrobial susceptibility testing for microbes that are difficult to grow represent the most immediate areas for application of WGS, and discuss the technical and infrastructure requirements for this to be implemented.
Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ.
Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs.
Researchers from Karlsruhe Institute of Technology (KIT) in Germany and the National Tsing Hua University in Taiwan have created a DNA-based (Salmon DNA-based memory device | KurzweilAI http://t.co/utx6zx9R...
Folding@home is a distributed computing project -- people from throughout the world download and run software to band together to make one of the largest supercomputers in the world. Every computer takes the project closer to our goals.
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