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Sequence library construction

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https://www.readbyqxmd.com/read/28727780/comparative-analysis-of-signature-genes-in-prrsv-infected-porcine-monocyte-derived-cells-to-different-stimuli
#1
Laura C Miller, Damarius S Fleming, Xiangdong Li, Darrell O Bayles, Frank Blecha, Yongming Sang
Monocyte-derived DCs (mDCs) are major target cells in porcine reproductive and respiratory syndrome virus (PRRSV) pathogenesis; however, the plasticity of mDCs in response to activation stimuli and PRRSV infection remains unstudied. In this study, we polarized mDCs, and applied genome-wide transcriptomic analysis and predicted protein-protein interaction networks to compare signature genes involved in mDCs activation and response to PRRSV infection. Porcine mDCs were polarized with mediators for 30 hours, then mock-infected, infected with PRRSV strain VR2332, or a highly pathogenic PRRSV strain (rJXwn06), for 5 h...
2017: PloS One
https://www.readbyqxmd.com/read/28726847/mapping-genome-wide-transcription-factor-binding-sites-using-dap-seq
#2
Anna Bartlett, Ronan C O'Malley, Shao-Shan Carol Huang, Mary Galli, Joseph R Nery, Andrea Gallavotti, Joseph R Ecker
To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX)...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726294/screening-and-structural-and-functional-investigation-of-a-novel-ferritin-from-phascolosoma-esculenta
#3
Hongwei Ding, Dijun Zhang, Shuangshuang Chu, Jun Zhou, Xiurong Su
Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two-hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co-immunoprecipitation. The pET-28a-FER147 prokaryotic expression vector was constructed...
July 20, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28724074/high-resolution-genetic-and-physical-mapping-of-the-eastern-filbert-blight-resistance-region-in-jefferson-hazelnut-corylus-avellana-l
#4
Vidyasagar Sathuvalli, Shawn A Mehlenbacher, David C Smith
Eastern filbert blight (EFB), caused by the pyrenomycete (Peck) E. Müller, is a devastating disease of European hazelnut ( L) in the US Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer 'Gasaway' confers a high level of resistance to EFB. To identify the gene responsible for resistance, we initiated map-based cloning efforts in a population of 1488 seedlings that segregated for resistance. Chromosome walking was initiated using primers designed from eight previously identified random amplified polymorphic DNA markers linked to resistance...
July 2017: Plant Genome
https://www.readbyqxmd.com/read/28723968/new-library-construction-method-for-single-cell-genomes
#5
Larry Xi, Alexander Belyaev, Sandra Spurgeon, Xiaohui Wang, Haibiao Gong, Robert Aboukhalil, Richard Fekete
A central challenge in sequencing single-cell genomes is the accurate determination of point mutations, phasing of these mutations, and identifying copy number variations with few assumptions. Ideally, this is accomplished under as low sequencing coverage as possible. Here we report our attempt to meet these goals with a novel library construction and library amplification methodology. In our approach, single-cell genomic DNA is first fragmented with saturated transposition to make a primary library that uniformly covers the whole genome by short fragments...
2017: PloS One
https://www.readbyqxmd.com/read/28713847/next-generation-sequencing-of-the-dna-virome-from-fecal-samples
#6
Cynthia L Monaco, Douglas S Kwon
Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified DNA is multiple-strand displacement amplified (MDA) and fragmented; and 4) libraries are constructed and sequenced using Illumina Miseq. Subsequent sequence analysis for viral sequence identification should be sensitive but stringent...
March 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28707884/rapid-and-programmable-protein-mutagenesis-using-plasmid-recombineering
#7
Sean Andrew Higgins, Sorel Ounkap, David F Savage
Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space...
July 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28705785/determination-of-certain-bacterial-groups-in-gut-microbiota-and-endotoxin-levels-in-patients-with-nonalcoholic-steatohepatitis
#8
Ceren Özkul, Meltem Yalınay, Tarkan Karakan, Güldal Yılmaz
BACKGROUND/AIMS: In recent years, the role of the gut microbiota has emerged in several diseases. Herein we aimed to determine the fecal microbiota, endotoxin levels, and inflammation markers in patients with nonalcoholic steatohepatitis (NASH) and healthy controls. MATERIALS AND METHODS: A total of 46 NASH patients and 38 healthy controls were included. NASH patients were diagnosed according to the steatosis, activity, and fibrosis score/fatty liver inhibition of progression algorithm...
July 13, 2017: Turkish Journal of Gastroenterology: the Official Journal of Turkish Society of Gastroenterology
https://www.readbyqxmd.com/read/28697453/electricity-production-and-microbial-characterization-of-thermophilic-microbial-fuel-cells
#9
Kun Dai, Jun-Li Wen, Fang Zhang, Xi-Wen Ma, Xiang-Yu Cui, Qi Zhang, Ting-Jia Zhao, Raymond J Zeng
Thermophilic microbial fuel cell (TMFC) offers many benefits, but the investigations on the diversity of exoelectrogenic bacteria are scarce. In this study, a two-chamber TMFC was constructed using ethanol as an electron donor, and the microbial dynamics were analyzed by high-throughput sequencing and 16S rRNA clone-library sequencing. The open-circuit potential of TMFC was approximately 650mV, while the maximum voltage was around 550mV. The maximum power density was 437mW/m(2), and the columbic efficiency in this work was 20...
July 1, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28689267/the-novel-srna-s015-improves-nisin-yield-by-increasing-acid-tolerance-of-lactococcus-lactis-f44
#10
Jiakun Qi, Qinggele Caiyin, Hao Wu, Kairen Tian, Binbin Wang, Yanni Li, Jianjun Qiao
Nisin, a polycyclic antibacterial peptide produced by Lactococcus lactis, is stable at low pH. Improving the acid tolerance of L. lactis could thus enhance nisin yield. Small non-coding RNAs (sRNAs) play essential roles in acid tolerance by regulating their target mRNAs at the post-transcriptional level. In this study, a novel sRNA, s015, was identified in L. lactis F44 via the use of RNA sequencing, qRT-PCR analysis, and Northern blotting. s015 improved the acid tolerance of L. lactis and boosted nisin yield at low pH...
July 9, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28684723/screening-and-validation-of-highly-efficient-insecticidal-conotoxins-from-a-transcriptome-based-dataset-of-chinese-tubular-cone-snail
#11
Bingmiao Gao, Chao Peng, Bo Lin, Qin Chen, Junqing Zhang, Qiong Shi
Most previous studies have focused on analgesic and anti-cancer activities for the conotoxins identified from piscivorous and molluscivorous cone snails, but little attention has been devoted to insecticidal activity of conotoxins from the dominant vermivorous species. As a representative vermivorous cone snail, the Chinese tubular cone snail (Conus betulinus) is the dominant Conus species inhabiting the South China Sea. We sequenced related venom transcriptomes from C. betulinus using both the next-generation sequencing and traditional Sanger sequencing technologies, and a comprehensive library of 215 conotoxin transcripts was constructed...
July 6, 2017: Toxins
https://www.readbyqxmd.com/read/28683952/identification-and-profile-of-micrornas-in-xiang-pig-testes-in-four-different-ages-detected-by-solexa-sequencing
#12
Zhi-Yu Luo, Xin-Lan Dai, Xue-Qin Ran, Yong-Xiu Cen, Xi Niu, Shi-Hui Huang, Jia-Fu Wang
To further understand the role of microRNA (miRNA) during testicular development, we constructed four small RNA libraries from the testes of the Chinese indigenous Xiang pig at four different ages, which were sequenced using high-throughput Solexa deep sequencing methods. It yielded over 23 million high-quality reads and 1,342,579 unique sequences. At two and three months of age, the proportion which represented miRNAs was the most abundant class of small RNAs, but it was gradually replaced by the category that represented piRNAs in adult testes...
June 28, 2017: Theriogenology
https://www.readbyqxmd.com/read/28681105/chimeras-linked-to-tandem-repeats-and-transposable-elements-in-tetraploid-hybrid-fish
#13
Lihai Ye, Ni Jiao, Xiaojun Tang, Yiyi Chen, Xiaolan Ye, Li Ren, Fangzhou Hu, Shi Wang, Ming Wen, Chun Zhang, Min Tao, Shaojun Liu
The formation of the allotetraploid hybrid lineage (4nAT) encompasses both distant hybridization and polyploidization processes. The allotetraploid offspring have two sets of sub-genomes inherited from both parental species, and therefore, it is important to explore its genetic structure. Herein, we construct a bacterial artificial chromosome library of allotetraploids, and then sequence and analyze the full-length sequences of 19 bacterial artificial chromosomes. Sixty-eight DNA chimeras are identified, which are divided into four models according to the distribution of the genomic DNA derived from the parents...
July 5, 2017: Marine Biotechnology
https://www.readbyqxmd.com/read/28680431/transcriptional-profiling-and-identification-of-heat-responsive-genes-in-perennial-ryegrass-by-rna-sequencing
#14
Kehua Wang, Yanrong Liu, Jinli Tian, Kunyong Huang, Tianran Shi, Xiaoxia Dai, Wanjun Zhang
Perennial ryegrass (Lolium perenne) is one of the most widely used forage and turf grasses in the world due to its desirable agronomic qualities. However, as a cool-season perennial grass species, high temperature is a major factor limiting its performance in warmer and transition regions. In this study, a de novo transcriptome was generated using a cDNA library constructed from perennial ryegrass leaves subjected to short-term heat stress treatment. Then the expression profiling and identification of perennial ryegrass heat response genes by digital gene expression analyses was performed...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28679365/increasing-quality-throughput-and-speed-of-sample-preparation-for-strand-specific-messenger-rna-sequencing
#15
Simon Haile, Richard D Corbett, Tina MacLeod, Steve Bilobram, Duane Smailus, Philip Tsao, Heather Kirk, Helen McDonald, Pawan Pandoh, Miruna Bala, Martin Hirst, Diane Miller, Richard A Moore, Andrew J Mungall, Jacquie Schein, Robin J Coope, Yussanne Ma, Yongjun Zhao, Rob A Holt, Steven J Jones, Marco A Marra
BACKGROUND: RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. METHODS: Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction...
July 5, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28674802/construction-and-application-of-random-dsrna-interference-library-for-functional-genetic-screens-in-embryonic-stem-cells
#16
Xiaoxing Cheng, Rui Jian
RNA interference (RNAi) libraries have been proven to be a powerful tool for large-scale functional genetic screens. To facilitate high-throughput functional genetic screens in embryonic stem cells, a system for construction of random dsRNA-expressing RNAi libraries was developed. Previous studies have demonstrated that sequence-specific gene silencing could be induced by long dsRNA in mouse embryos, mouse oocytes, embryonic stem cells, and some other mammalian cells. Our study demonstrated that the dsRNA interference library can be used for functional genetic screens of genes involved in self-renewal of embryonic stem cells (ES cells)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28674067/genome-scale-mutational-analysis-of-geobacter-sulfurreducens-reveals-distinct-molecular-mechanisms-for-respiration-and-sensing-of-poised-electrodes-vs-fe-iii-oxides
#17
Chi Ho Chan, Caleb E Levar, Fernanda Jiménez-Otero, Daniel R Bond
Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth with soluble fumarate or an electrode as the electron acceptor...
July 3, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28668702/differential-expression-of-novel-micrornas-in-response-to-the-infection-of-a-tmv-mutant-with-an-internal-poly-a-tract-in-n-benthamiana
#18
Song Guo, Ya-Chih Hsueh, Greg Tucker-Kellogg, Sek-Man Wong
We first constructed small RNA libraries of TMV- and TMV-43A-infected N. benthamiana for high throughput sequencing. A total number of 181 novel microRNAs (miRNAs) were identified through an improved miRNAs analysis pipeline. We were able to identify consistent miRNA expression changes induced in TMV and TMV-43A-infected plants, as well as differences associated with the UPD substitution in the TMV-43A viral genome. Virally induced miRNAs are associated with distinct processes and functions of predicted mRNA targets, including relation to host target defense...
June 28, 2017: Virus Research
https://www.readbyqxmd.com/read/28662150/a-viral-metagenomic-approach-on-a-non-metagenomic-experiment-mining-next-generation-sequencing-datasets-from-pig-dna-identified-several-porcine-parvoviruses-for-a-retrospective-evaluation-of-viral-infections
#19
Samuele Bovo, Gianluca Mazzoni, Anisa Ribani, Valerio Joe Utzeri, Francesca Bertolini, Giuseppina Schiavo, Luca Fontanesi
Shot-gun next generation sequencing (NGS) on whole DNA extracted from specimens collected from mammals often produces reads that are not mapped (i.e. unmapped reads) on the host reference genome and that are usually discarded as by-products of the experiments. In this study, we mined Ion Torrent reads obtained by sequencing DNA isolated from archived blood samples collected from 100 performance tested Italian Large White pigs. Two reduced representation libraries were prepared from two DNA pools constructed each from 50 equimolar DNA samples...
2017: PloS One
https://www.readbyqxmd.com/read/28654052/flow-sorting-and-exome-sequencing-of-the-reed-sternberg-cells-of-classical-hodgkin-lymphoma
#20
Jonathan B Reichel, Jason McCormick, Jonathan R Fromm, Olivier Elemento, Ethel Cesarman, Mikhail Roshal
The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study...
June 10, 2017: Journal of Visualized Experiments: JoVE
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