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Stéphanie Raymond, Florence Nicot, Romain Carcenac, Caroline Lefebvre, Nicolas Jeanne, Karine Saune, Pierre Delobel, Jacques Izopet
Objectives: To evaluate the diagnostic performance of the Vela next-generation sequencing (NGS) system in conjunction with the Sentosa SQ HIV Genotyping Assay for genotyping HIV-1. Methods: Plasma RNA was extracted and templates prepared with the Sentosa SX instrument before sequencing the HIV-1 polymerase on the Sentosa SQ301 Sequencer (PGM IonTorrent). The Vela NGS System was compared with direct sequencing and the 454 GS-FLX (Roche) and MiSeq (Illumina) systems for genotypic resistance testing on clinical samples...
February 12, 2018: Journal of Antimicrobial Chemotherapy
Wai-Sing Chan, Chun-Hang Au, Dona N Ho, Tsun-Leung Chan, Edmond Shiu-Kwan Ma, Bone Siu-Fai Tang
BACKGROUND: Human fecal carriage of Enterobacteriaceae possessing mobilized colistin resistance genes (mcr-1 and mcr-2) remains obscure in Hong Kong. As part of routine surveillance on emerging antibiotic resistance, we conducted a prospective study on this topic in a regional hospital in Hong Kong. METHODS: From October 31 to November 25, 2016, all fecal specimens submitted for routine analysis were included in this surveillance study. These comprised 672 consecutive routine fecal specimens collected from 616 individuals...
February 13, 2018: BMC Infectious Diseases
Silje L Rekdal, Jarl Andreas Anmarkrud, Arild Johnsen, Jan T Lifjeld
Genotyping of classical major histocompatibility complex (MHC) genes is challenging when they are hypervariable and occur in multiple copies. In this study, we used several different approaches to genotype the moderately variable MHC class I exon 3 (MHCIe3) and the highly polymorphic MHC class II exon 2 (MHCIIβe2) in the bluethroat (Luscinia svecica). Two family groups (eight individuals) were sequenced in replicates at both markers using Ion Torrent technology with both a single- and a dual-indexed primer structure...
February 2018: Ecology and Evolution
Neta Luria, Elisheva Smith, Noa Sela, Oded Lachman, Ilana Bekelman, Amnon Koren, Aviv Dombrovsky
During October 2014, unfamiliar mild mosaic and mottling symptoms were identified on leaves of pepper (Capsicum chinense cv. Habanero) seedlings grown in the Arava valley in Israel 2-3 weeks post planting. Symptomatic plants were tested positive by ELISA using laboratory-produced antisera for tobamovirus species. Typical tobamovirus rod-shaped morphology was observed by transmission electron microscopy (TEM) analysis of purified virion preparation that was used for mechanical inoculation of laboratory test plants for the completion of Koch's postulates...
February 10, 2018: Virus Genes
E Humer, S Aditya, A Kaltenegger, F Klevenhusen, R M Petri, Q Zebeli
A new segment of feed industry based on bakery by-products (BBP) has emerged. Yet, information is lacking regarding the effects of inclusion of BBP in ruminant diets on ruminal fermentation and microbiota. Therefore, the aim of this study was to evaluate the effect of the gradual replacement of grains by BBP on ruminal fermentation, nutrient degradation, and microbial community composition using the rumen-simulation technique. All diets consisted of hay and concentrate mixture with a ratio of 42:58 (dry matter basis), but differed in the concentrate composition with either 45% cereal grains or BBP, whereby 15, 30, or 45% of BBP were used in place of cereal grains...
February 7, 2018: Journal of Dairy Science
Anne Bergougnoux, Valeria D'Argenio, Stefanie Sollfrank, Fanny Verneau, Antonella Telese, Irene Postiglione, Karl J Lackner, Mireille Claustres, Giuseppe Castaldo, Heidi Rossman, Francesco Salvatore, Caroline Raynal
BACKGROUND: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom's CFTR MASTR Dx kit to obtain CE-IVD certification...
February 10, 2018: Clinical Chemistry and Laboratory Medicine: CCLM
Jun Zheng, Jun-Qiao Feng, Lei Zhou, Serge Maurice Mbadinga, Ji-Dong Gu, Bo-Zhong Mu
Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents...
February 9, 2018: World Journal of Microbiology & Biotechnology
Jing Yuan, Yongkun Yuan, Yihang Zhu, Linkui Cao
Paddy soil accounts for 10% of global atmospheric methane (CH4) emissions. Many types of fertilizers may enhance CH4 emissions, especially organic fertilizer. The aim of this study was to explore the effects of different fertilizers on CH4 and methanogen patterns in paddy soil. This experiment involved four treatments: chemical fertilizer (CT), organic fertilizer (OT), mixed with chemical and organic fertilizer (MT), and no fertilizer (ctrl). The three fertilization treatments were applied with total nitrogen at the same rate of 300 kg N ha-1...
February 1, 2018: Science of the Total Environment
Lan Wang, Jing Zhang, Huilin Li, Hong Yang, Chao Peng, Zhengsong Peng, Lu Lu
Urban rivers represent a unique ecosystem in which pollution occurs regularly, leading to significantly altered of chemical and biological characteristics of the surface water and sediments. However, the impact of urbanization on the diversity and structure of the river microbial community has not been well documented. As a major tributary of the Yangtze River, the Jialing River flows through many cities. Here, a comprehensive analysis of the spatial microbial distribution in the surface water and sediments in the Nanchong section of Jialing River and its two urban branches was conducted using 16S rRNA gene-based Illumina MiSeq sequencing...
January 30, 2018: Science of the Total Environment
Piers Blombery, Kate Jones, Ken Doig, Georgina Ryland, Michelle McBean, Ella Thompson, Costas K Yannakou, David Westerman
CONTEXT: - Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. OBJECTIVE: - To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×-500 000×) sequencing (NGS-q NPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. DESIGN: - A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house-designed bioinformatic pipeline...
February 9, 2018: Archives of Pathology & Laboratory Medicine
Rupesh Kanchi Ravi, Kendra Walton, Mahdieh Khosroheidari
MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1 × 36 base pairs to 2 × 300 base pairs...
2018: Methods in Molecular Biology
Yu-Ling Sun, Wen-Qi Li, Peng-Xu Ding, Zhi-Wei Wang, Chang-Hua Wei, Xiu-Xian Ma, Rui-Fang Zhang, Yan Wu, Lin Zhou, Ruo-Peng Liang, Yan-Peng Zhang, Yi-Pu Zhao, Rong-Tao Zhu, Jian Li
Gut microbiota is associated with liver diseases. However, gut microbial characteristics of Budd-Chiari syndrome (B-CS) have not been reported. Here, by MiSeq sequencing, gut microbial alterations were characterized among 37 health controls, 20 liver cirrhosis (LC) patients, 31 initial B-CS patients (B-CS group), 33 stability patients after BCS treatment (stability group) and 23 recurrent patients after BCS treatment (recurrence group). Gut microbial diversity was increased in B-CS versus LC. Bacterial community of B-CS clustered with controls but separated from LC...
January 9, 2018: Oncotarget
Silvana Teresa Tapia-Paniagua, Diana Ceballos-Francisco, Ma Carmen Balebona, María Ángeles Esteban, Miguel Ángel Moriñigo
Interest in fish skin immunity and its associated microbiota has greatly increased among immunologists. The objective of this study is to know if skin ulcers may be associated with changes in the mucus composition and microbial diversity. The abundance of terminal carbohydrates, several enzymes (protease, antiprotease, peroxidase, lysozyme) and total immunoglobulin M levels were evaluated in skin mucus of experimentally ulcered gilthead seabream (Sparus aurata L.). Furthermore, the composition of the microbiota of ulcered and non-ulcered skin has been determined using Illumina Miseq technology...
February 5, 2018: Fish & Shellfish Immunology
Stefanie U Wetzels, Melanie Eger, Marion Burmester, Lothar Kreienbrock, Amir Abdulmawjood, Beate Pinior, Martin Wagner, Gerhard Breves, Evelyne Mann
The rumen simulation technique (RUSITEC) is a well-established semicontinuous in vitro model for investigating ruminal fermentation; however, information on the stability of the ruminal bacterial microbiota and metabolome in the RUSITEC system is rarely available. The availability of high resolution methods, such as high-throughput sequencing and metabolomics improve our knowledge about the rumen microbial ecosystem and its fermentation processes. Thus, we used Illumina MiSeq 16S rRNA amplicon sequencing and a combination of direct injection mass spectrometry with a reverse-phase LC-MS/MS to evaluate the dynamics of the bacterial community and the concentration of several metabolites in a RUSITEC experiment as a function of time and in response to a challenge with a pathogenic Clostridium perfringens (C...
2018: PloS One
Laurence Devesse, David Ballard, Lucinda Davenport, Immy Riethorst, Gabriella Mason-Buck, Denise Syndercombe Court
By using sequencing technology to genotype loci of forensic interest it is possible to simultaneously target autosomal, X and Y STRs as well as identity, ancestry and phenotypic informative SNPs, resulting in a breadth of data obtained from a single run that is considerable when compared to that generated with standard technologies. It is important however that this information aligns with the genotype data currently obtained using commercially available kits for CE-based investigations such that results are compatible with existing databases and hence can be of use to the forensic community...
November 4, 2017: Forensic Science International. Genetics
Michelle A Peck, Kimberly Sturk-Andreaggi, Jacqueline T Thomas, Robert S Oliver, Suzanne Barritt-Ross, Charla Marshall
Generating mitochondrial genome (mitogenome) data from reference samples in a rapid and efficient manner is critical to harnessing the greater power of discrimination of the entire mitochondrial DNA (mtDNA) marker. The method of long-range target enrichment, Nextera XT library preparation, and Illumina sequencing on the MiSeq is a well-established technique for generating mitogenome data from high-quality samples. To this end, a validation was conducted for this mitogenome method processing up to 24 samples simultaneously along with analysis in the CLC Genomics Workbench and utilizing the AQME (AFDIL-QIAGEN mtDNA Expert) tool to generate forensic profiles...
January 16, 2018: Forensic Science International. Genetics
Gui-Hua Lu, Cheng-Yi Tang, Xiao-Mei Hua, Jing Cheng, Gu-Hao Wang, Yin-Ling Zhu, Li-Ya Zhang, Hui-Xia Shou, Jin-Liang Qi, Yong-Hua Yang
The increased worldwide commercial cultivation of transgenic crops during the past 20 years is accompanied with potential effects on the soil microbial communities, because many rhizosphere and endosphere bacteria play important roles in promoting plant health and growth. Previous studies reported that transgenic plants exert differential effects on soil microbial communities, especially rhizobacteria. Thus, this study compared the soybean root-associated bacterial communities between a 5-enolpyruvylshikimate-3-phosphate synthase -transgenic soybean line (ZUTS31 or simply Z31) and its recipient cultivar (Huachun3 or simply HC3) at the vegetative, flowering, and seed-filling stages...
2018: PloS One
Soo Jin Jeon, Fabio S Lima, Achilles Vieira-Neto, Vinicius S Machado, Svetlana F Lima, Rodrigo C Bicalho, Jose Eduardo P Santos, Klibs N Galvão
Broad-spectrum antibiotics such as ceftiofur and ampicillin are recommended for the treatment of metritis in dairy cows. Nonetheless, little is known about the impacts of antibiotics on the uterine microbiota. Here, we evaluated the shift in uterine microbiota after treating metritic cows with ceftiofur or ampicillin, and also gained insight into the uterine microbiota associated with cure of metritis. Uterine swabs from ceftiofur-treated, ampicillin-treated, and untreated metritic Holstein cows were collected on the day of metritis diagnosis (D1) and on D6 and then used for genomic DNA extraction and sequencing of the V4 hypervariable region of the 16S rRNA gene on the Illumina MiSeq platform...
February 2018: Veterinary Microbiology
Miaochun Fan, Xiao Xiao, Yanqing Guo, Jun Zhang, Entao Wang, Weimin Chen, Yanbing Lin, Gehong Wei
Heavy metals can cause serious contamination of soils, especially in mining regions. A detailed understanding of the effects of heavy metals on plants and root-associated microbial communities could help to improve phytoremediation systems. In this study, black locust (Robinia pseudoacacia) seedlings with or without rhizobial inoculation were planted in soils contaminated with different levels of heavy metals. Bacterial communities in rhizosphere and bulk soil samples were analyzed using 16S rRNA gene sequencing on the Illumina MiSeq platform and shotgun metagenome sequencing on the Illumina HiSeq platform...
February 2, 2018: Chemosphere
Xin Liu, Takashi Kuda, Hajime Takahashi, Bon Kimura
The Rubing milk cake from Yunnan and the Yan-cai vegetable pickles from Guangdong are traditional spontaneously fermented foods in China. We evaluated the microbial properties of these products with the analysis of their bacterial and fungal microbiota using classical culture-dependent and culture-independent methods, including a 16S rDNA gene (V4) and an internal transcribed spacer (ITS) region pyrosequencing method with MiSeq system. The viable lactic acid bacteria (LAB) count was 8 and 6 log colony-forming units (CFU)/g in Rubing and Yan-cai samples, respectively...
June 2018: Food Microbiology
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