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Sequence library construction

Shengru Wu, Jiacheng Guo, Liqin Zhu, Jiantao Yang, Si Chen, Xiaojun Yang
1. There has been substantial research focused on the roles of microRNAs (miRNA) and PIWI-interacting RNAs (piRNA) delivered from mammalian spermatozoa, however comparatively little is known about the role of spermatozoa-delivered miRNAs and piRNAs within breeding cockerels' spermatozoa. 2. A small RNA library of cockerels' spermatozoa was constructed using Illumina high-throughput sequencing technology. Unique sequences with lengths of 18-26 nucleotides were mapped to miRBase 21.0 and unique sequences with lengths of 25-37 nucleotides were mapped to a piRNA database...
April 18, 2018: British Poultry Science
Maikel Boot, Susanna Commandeur, Amit K Subudhi, Meriem Bahira, Trever C Smith, Abdallah M Abdallah, Mae van Gemert, Joël Lelièvre, Lluís Ballell, Bree B Aldridge, Arnab Pain, Alexander Speer, Wilbert Bitter
Due to the rise of drug resistant forms of tuberculosis there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life-death screening that give little qualitative information. In doing so, promising compound scaffolds or non-optimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early TB drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to sub-inhibitory concentrations of antibiotics with known targets: ciprofloxacin, ethambutol, isoniazid, streptomycin and rifampicin...
April 16, 2018: Antimicrobial Agents and Chemotherapy
Jinnan Wang, Yanting Tian, Jihong Li, Keqiang Yang, Shiyan Xing, Xiaojiao Han, Dong Xu, Yiwei Wang
Branching in woody plants affects their ecological benefits and impacts wood formation. To obtain genome-wide insights into the transcriptome changes and regulatory mechanisms associated with branching, we performed high-throughput RNA sequencing to characterize cDNA libraries generated from active buds of Populus deltoides CL. 'zhonglin2025' (BC) and Populus × zhaiguanheibaiyang (NC). NC has more branches than BC and rapid growth. We obtained a total of 198.2 million high-quality clean reads from the NC and BC libraries...
April 13, 2018: Genomics
Shruti Choudhary, Sapna Thakur, Raoof Ahmad Najar, Aasim Majeed, Amandeep Singh, Pankaj Bhardwaj
Rhododendron arboreum, an ecologically prominent species, also lends commercial and medicinal benefits in the form of palatable juices and useful herbal-drugs. The local abundance and survival of the species under highly fluctuating climate make it an ideal model for genetic structure and functional analysis. However, a lack of genomic data has hampered the additional research. In the present study, cDNA libraries from floral and foliar tissues of the species were sequenced to provide a foundation for understanding the functional aspects of the genome and to construct an enriched repository that will promote genomics studies in the genera...
April 15, 2018: Genome Génome / Conseil National de Recherches Canada
Andrew Lucas, Owen Bodger, Berry J Brosi, Col R Ford, Dan W Forman, Carolyn Greig, Matthew Hegarty, Penelope J Neyland, Natasha de Vere
Pollination by insects is a key ecosystem service and important to wider ecosystem function. Most species-level pollination networks studied have a generalised structure, with plants having several potential pollinators, and pollinators in turn visiting a number of different plant species. This is in apparent contrast to a plant's need for efficient conspecific pollen transfer. The aim of this study was to investigate the structure of pollen transport networks at three levels of biological hierarchy: community, species and individual...
April 16, 2018: Journal of Animal Ecology
Hushuai Nie, Yumei Wang, Ying Su, Jinping Hua
Cytoplasmic male sterility (CMS) lines provide crucial material to harness heterosis for crop plants, which serves as an important strategy for hybrid seed production. However, the molecular mechanism remains obscure. Although microRNAs (miRNAs) play important roles in vegetative growth and reproductive growth, there are few reports on miRNAs regulating the development of male sterility in Upland cotton. In present study, 12 small RNA libraries were constructed and sequenced for two development stages of flower buds from a CMS line and its maintainer line...
April 7, 2018: Functional & Integrative Genomics
Craig J van Dolleweerd, Sarah A Kessans, Kyle C Van de Bittner, Leyla Y Bustamante, Rudranuj Bundela, Barry Scott, Matthew J Nicholson, Emily J Parker
A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements...
April 5, 2018: ACS Synthetic Biology
Brandon T Gufford, Jason D Robarge, Michael T Eadon, Hongyu Gao, Hai Lin, Yunlong Liu, Zeruesenay Desta, Todd C Skaar
Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0...
April 2018: Pharmacology Research & Perspectives
Gilberto Santos de Oliveira, Rebeca Kawahara, Livia Rosa-Fernandes, Simon Ngao Mule, Carla Cristi Avila, Marta M G Teixeira, Martin R Larsen, Giuseppe Palmisano
BACKGROUND: Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features...
April 2, 2018: PLoS Neglected Tropical Diseases
Shuanghong Zhang, Dingyu Liu, Zhitao Mao, Yufeng Mao, Hongwu Ma, Tao Chen, Xueming Zhao, Zhiwen Wang
OBJECTIVE: To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum. RESULTS: A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the - 10 and - 35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model...
May 2018: Biotechnology Letters
Ying Zhao, Wengang Yu, Xiangyu Hu, Youhai Shi, Yu Liu, Yunfang Zhong, Peng Wang, Shuya Deng, Jun Niu, Xudong Yu
Molecular regulatory mechanism of heat stress response (HSR) in Ericaceae remains unknown. Here, we sought to identify HSR mechanisms in Rhododendron hainanense, a Ericaceae species, through a combination of physiological and transcriptomic studies. The levels of MDA, H2 O2 , Pro, SOD, CAT and APX in leaves of R. hainanense were analyzed to characterize a dramatic difference in varied temperature treatment. Also, three sequencing libraries, including one control and two heat stress (HS)-treated samples, were constructed for comparative transcriptomic analysis...
March 28, 2018: Gene
Rihab Ameri, Elisabeth Laville, Gabrielle Potocki-Véronèse, Sahar Trabelsi, Monia Mezghani, Fatma Elgharbi, Samir Bejar
Dromedaries are capable of digesting plant cell wall with high content of lignocellulose of poor digestibility. Consequently, their intestinal microbiota can be a source of novel carbohydrate-active enzymes (CAZymes). To the best of our knowledge, no data are available describing the biochemical analysis of enzymes in dromedary intestinal microbiota. To investigate new hydrolytic enzymes from the dromedary gut, a fosmid library was constructed using metagenomic DNA from feces of non-domestic adult dromedary camels living in the Tunisian desert...
2018: PloS One
Kathy N Lam, Eric C Martens, Trevor C Charles
Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters...
May 2018: MSystems
Marcella A Teixeira, Noa Sela, Hagop S Atamian, Ergude Bao, Ritu Chaudhary, Jacob MacWilliams, Jiangman He, Sophie Mantelin, Thomas Girke, Isgouhi Kaloshian
The potato aphid, Macrosiphum euphorbiae, is an important agricultural pest that causes economic losses to potato and tomato production. To establish the transcriptome for this aphid, RNA-Seq libraries constructed from aphids maintained on tomato plants were used in Illumina sequencing generating 52.6 million 75-105 bp paired-end reads. The reads were assembled using Velvet/Oases software with SEED preprocessing resulting in 22,137 contigs with an N50 value of 2,003bp. After removal of contigs from tomato host origin, 20,254 contigs were annotated using BLASTx searches against the non-redundant protein database from the National Center for Biotechnology Information (NCBI) as well as IntereProScan...
2018: PloS One
Tao Sun, Xue-Qing Wang, Zun-Ling Zhao, Shu-Hui Yu, Pu Yang, Xiao-Ming Chen
The Chinese white wax scale insect (Ericerus pela) is an economically valuable species with an important role in wax production. Recently, in a greenhouse in Kunming, we identified a genus of fungus that infects and kills E. pela females. This study sought to perform the molecular detection of entomopathogens and analyze the changes in the host microbiota after entomopathogen infection. We used library construction, high-throughput sequencing and real-time quantitative polymerase chain reaction (RT-qPCR) to identify the fungi infecting adult E...
March 28, 2018: Scientific Reports
Matthew T Aliota, Dawn M Dudley, Christina M Newman, James Weger-Lucarelli, Laurel M Stewart, Michelle R Koenig, Meghan E Breitbach, Andrea M Weiler, Matthew R Semler, Gabrielle L Barry, Katie R Zarbock, Amelia K Haj, Ryan V Moriarty, Mariel S Mohns, Emma L Mohr, Vanessa Venturi, Nancy Schultz-Darken, Eric Peterson, Wendy Newton, Michele L Schotzko, Heather A Simmons, Andres Mejia, Jennifer M Hayes, Saverio Capuano, Miles P Davenport, Thomas C Friedrich, Gregory D Ebel, Shelby L O'Connor, David H O'Connor
Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a "synthetic swarm" whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids...
March 28, 2018: PLoS Pathogens
Brett A Case, Max A Kruziki, Sadie M Johnson, Benjamin J Hackel
The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding...
March 26, 2018: Bioconjugate Chemistry
Saman Saeidi, Michael R McKain, Elizabeth A Kellogg
Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples...
March 8, 2018: Journal of Visualized Experiments: JoVE
Guodong Feng, Shengxiong Huang, Ying Liu, Fangming Xiao, Jian Liu, Zheng Zhang, Qiang Chen, Yun Mao, Xulv Cao, Yang Wang, Danyang Chen, Yu Zhou, Fengyuan Yu, Guoqing Liu, Yongsheng Liu, Xiangli Niu
Genomic and transcriptomic sequences of Tagetes erecta are very limited in public databases, despite its nutritional and economical value. In this study, nine cDNA libraries were constructed from leaves, immature and mature flowers and sequenced using Illumina Hiseq 2000. Over 45.66 Gb nucleotides were generated and a total of 72,499 unigenes were assembled, 34,892 (48.13%) of which were annotated in NR, Swiss-prot, COG, GO, KOG, KEGG and Pfam. 11,721 differentially expressed genes were identified in leaves, immature flowers and mature flowers...
March 21, 2018: Gene
Amanuel Menghs Ghilamicael, Hamadi Iddi Boga, Sylvester Elikana Anami, Tadesse Mehari, Nancy L M Budambula
Human pathogens can survive and grow in hot springs. For water quality assessment, Escherichia coli or Enterococci are the main thermotolerant enteric bacteria commonly used to estimate the load of pathogenic bacteria in water. However, most of the environmental bacteria are unculturable thus culture methods may cause bias in detection of most pathogens. Illumina sequencing can provide a more comprehensive and accurate insight into environmental bacterial pathogens, which can be used to develop better risk assessment methods and promote public health awareness...
2018: PloS One
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