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Sequence library construction

Emily K Fowler, Irina Mohorianu, Damian T Smith, Tamas Dalmay, Tracey Chapman
RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions...
2018: PloS One
Muhammad Shakeel, Xiaoxia Xu, Jin Xu, Shuzhong Li, Jialin Yu, Xianqiang Zhou, Xiaojing Xu, Qiongbo Hu, Xiaoqiang Yu, Fengliang Jin
Plutella xylostella , a global key pest, is one of the major lepidopteran pests of cruciferous vegetables owing to its strong ability of resistance development to a wide range of insecticides. Destruxin A, a mycotoxin of the entomopathogenic fungus, Metarhizium anisopliae , has broad-spectrum insecticidal effects and has been used as an alternative control strategy to reduce harmful effects of insecticides. However, microRNA (miRNA)-regulated reactions against destruxin A have not been elucidated yet. Therefore, here, to identify immunity-related miRNAs, we constructed four small RNA libraries from destruxin A-injected larvae of P...
2018: Frontiers in Immunology
P San Segundo-Acosta, M Garranzo-Asensio, C Oeo-Santos, A Montero-Calle, J Quiralte, J Cuesta-Herranz, M Villalba, R Barderas
Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning...
February 19, 2018: Journal of Immunological Methods
Emily C A Goodall, Ashley Robinson, Iain G Johnston, Sara Jabbari, Keith A Turner, Adam F Cunningham, Peter A Lund, Jeffrey A Cole, Ian R Henderson
Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12...
February 20, 2018: MBio
Jian Wu, Wei Dai, Lin Wu, Jinke Wang
BACKGROUND: Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. RESULTS: This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA)...
February 13, 2018: BMC Genomics
Yifeng Zhou, Qiaoyu Tang, Meiru Wu, Di Mou, Hui Liu, Shouchuang Wang, Chi Zhang, Li Ding, Jie Luo
Selenium (Se) is an essential mineral element for animals and humans. Cardamine hupingshanensis (Brassicaceae), found in the Wuling mountain area of China, has been identified as a novel Se hyperaccumulator plant. However, the mechanism for selenium tolerance in Cardamine plants remains unknown. In this study, two cDNA libraries were constructed from seedlings of C. hupingshanensis treated with selenite. Approximately 100 million clean sequencing reads were de novo assembled into 48,989 unigenes, of which 39,579 and 33,510 were expressed in the roots and leaves, respectively...
February 12, 2018: Scientific Reports
Daniel J Gates, Diana Pilson, Stacey D Smith
Advances in sequencing technology have made it possible to produce large multi-locus datasets required for species tree analyses. One challenge with constructing high throughput sequencing datasets, however, is that missing information is propagated at different steps in the sequence preparation process. To date, species tree studies have focused on filtering and removing errors that occur at particular loci. Given the way that high throughput sequencing datasets are constructed, however, large amounts of error or ambiguity may also manifest across individuals...
February 9, 2018: Molecular Phylogenetics and Evolution
Xiao-Hong Li, Ricardo DeMarco, Leandro X Neves, Sally R James, Katherine Newling, Peter D Ashton, Jian-Ping Cao, R Alan Wilson, William Castro-Borges
BACKGROUND: Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death...
February 12, 2018: PLoS Neglected Tropical Diseases
Winnie S Liang, Kristi Stephenson, Jonathan Adkins, Austin Christofferson, Adrienne Helland, Lori Cuyugan, Jonathan J Keats
Whole exome sequencing (WES) is a DNA sequencing strategy that provides a survey of base substitutions across coding genomic locations and other regions of interest. As the coding portion of the genome encompasses only 1-2% of the entire genome, this approach represents a more cost-effective strategy to detect DNA alterations that may alter protein function, compared to whole genome sequencing. Although the research community has and is currently delineating the functional implications of sequence changes in noncoding regions of the genome, WES is a currently available assay that provides valuable information for both discovery research and precision medicine applications...
2018: Methods in Molecular Biology
Jonathan J Keats, Lori Cuyugan, Jonathan Adkins, Winnie S Liang
With the rapid evolution of genomics technologies over the past decade, whole genome sequencing (WGS) has become an increasingly accessible tool in biomedical research. WGS applications include analysis of genomic DNA from single individuals, multiple related family members, and tumor/normal samples from the same patient in the context of oncology. A number of different modalities are available for performing WGS; this chapter focuses on wet lab library construction procedures for complex short insert WGS libraries using the KAPA Hyper Prep Kit (Kapa Biosystems), and includes a discussion of appropriate quality control measures for sequencing on the Illumina HiSeq2000 platform...
2018: Methods in Molecular Biology
Arie Ryvkin, Haim Ashkenazy, Yael Weiss-Ottolenghi, Chen Piller, Tal Pupko, Jonathan M Gershoni
Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases...
February 6, 2018: Nucleic Acids Research
Vicente Pérez-Brocal, Andrés Moya
We have analysed oral wash samples from 72 healthy young adults in Valencia (Spain) for a metagenomic analysis through the construction of shotgun libraries and high-throughput-sequencing. The oral viral communities have been taxonomically characterised as well as and the gene content from the latter. The majority of viruses are found in few individuals, with single occurrences being the most widespread ones, whereas universally distributed viruses, while present, are relatively rare, with bacteriophages from families Siphoviridae and Myoviridae, and Streptococcus phages, as well as the eukaryotic viral family Herpesviridae amongst the most widespread viruses...
2018: PloS One
Chunsun Gu, Sheng Xu, Zhiquan Wang, Liangqin Liu, Yongxia Zhang, Yanming Deng, Suzhen Huang
As a halophyte, Iris lactea var. chinensis (I. lactea var. chinensis) is widely distributed and has good drought and heavy metal resistance. Moreover, it is an excellent ornamental plant. I. lactea var. chinensis has extensive application prospects owing to the global impacts of salinization. To better understand its molecular mechanism involved in salt resistance, the de novo sequencing, assembly, and analysis of I. lactea var. chinensis roots' transcriptome in response to salt-stress conditions was performed...
January 27, 2018: Plant Physiology and Biochemistry: PPB
Grant S Schulert, Mingce Zhang, Ammar Husami, Ndate Fall, Hermine Brunner, Kejian Zhang, Randy Q Cron, Alexei A Grom
OBJECTIVE: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (SJIA), and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH-3 (FHL3), but the role of non-coding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. METHODS: A custom enrichment library was constructed to sequence approximately 1 MB flanking UNC13D in 24 patients with SJIA, recurrent MAS, and negative prior genetic (exon/coding) testing...
February 6, 2018: Arthritis & Rheumatology
Chao Wang, Shuang Chen, Hong-Bin Zhang, Yao Li, Xue-Qin Hu
Dextran produced by dextransucrase hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown. Previous studies have investigated the relationships between structure and linkage specificity of the dextransucrase DSR from Leuconostoc mesenteroides by the site-directed mutagenesis of the catalytic pocket. The glycosidic linkage of dextran produced by mutant enzymes changed slightly by 3% to 20%. The mutagenesis dextransucrases were constructed by inserting an amino acid into a catalytic pocket to investigate the product specificities of dextransucrase thoroughly...
February 2, 2018: International Journal of Biological Macromolecules
Koeun Han, Hea-Young Lee, Na-Young Ro, On-Sook Hur, Joung-Ho Lee, Jin-Kyung Kwon, Byoung-Cheorl Kang
Capsaicinoids are unique compounds produced only in peppers (Capsicum spp.). Several studies using classical quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS) have identified QTLs controlling capsaicinoid content in peppers; however, neither the QTLs common to each population nor the candidate genes underlying them have been identified due to the limitations of each approach used. Here, we performed QTL mapping and GWAS for capsaicinoid content in peppers using two recombinant inbred line (RIL) populations and one GWAS population...
February 6, 2018: Plant Biotechnology Journal
Jingjing Gong, Xiaoyan Du, Zhenkun Li, Xiaohong Li, Meng Guo, Jing Lu, Ying Wang, Zhenwen Chen, Changlong Li
OBJECTIVES: We aimed at identifying genes related to hereditary type 2 diabetes expressed in the liver and the adipose tissue of spontaneous diabetic gerbils using suppression subtractive hybridization (SSH) screening. METHODS: Two gerbil littermates, one with high and the other with normal blood glucose level, from our previously bred spontaneous diabetic gerbil strain were used in this study. To identify differentially expressed genes in the liver and the adipose tissue, mRNA from these tissues was extracted and SSH libraries were constructed for screening...
2018: PloS One
Gaurang G Gowande, Sanket Tembe, Hemant V Ghate
Cytochrome c oxidase subunit I gene (COI) sequences of roughly 509 bp length for various species of the Infraorder Pentatomomorpha were generated. K2P divergences within and between species and genera were calculated and compared using newly generated sequences and the ones available on online portals. Mean interspecific (within-genus) genetic divergence (14.23%) was ∼ eight times greater than mean intraspecific (within-species) divergence (1.79%). Distance-based as well as character-based approaches were used towards constructing (COI) trees...
February 1, 2018: Mitochondrial DNA. Part A. DNA Mapping, Sequencing, and Analysis
Zhaofeng Wang, Ana G Herraiz, Ana M Del Hoyo, Marcos G Suero
Carbon has the unique ability to bind four atoms and form stable tetravalent structures that are prevalent in nature. The lack of one or two valences leads to a set of species-carbocations, carbanions, radicals and carbenes-that is fundamental to our understanding of chemical reactivity. In contrast, the carbyne-a monovalent carbon with three non-bonded electrons-is a relatively unexplored reactive intermediate; the design of reactions involving a carbyne is limited by challenges associated with controlling its extreme reactivity and the lack of efficient sources...
January 31, 2018: Nature
Guillaume Urtecho, Arielle D Tripp, Kimberly Insigne, Hwangbeom Kim, Sriram Kosuri
Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In E. coli , decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange (RMCE) system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations...
February 1, 2018: Biochemistry
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