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Sequence library construction

Xin-Yu Chen, Yang Yang, Li-Ping Ran, Zhao-di Dong, Er-Jin Zhang, Xu-Run Yu, Fei Xiong
Drought stress is a significant abiotic stress factor that affects wheat yield and quality. MicroRNA (miRNA) plays an important role in regulating caryopsis development in response to drought stress. However, little is known about the expression characteristics of miRNAs and how they regulate protein accumulation in wheat caryopsis under drought stress. To address this, two small RNA libraries of wheat caryopsis under control and drought stress conditions were constructed and sequenced. A total of 125 miRNAs were identified in the two samples, of which 110 were known and 15 were novel...
2017: Frontiers in Plant Science
Yingxin Zhang, Chengming Fan, Shuangshuang Li, Yuhong Chen, Richard R-C Wang, Xiangqi Zhang, Fangpu Han, Zanmin Hu
Transposable elements (TEs) in plant genomes exhibit a great variety of structure, sequence content and copy number, making them important drivers for species diversity and genome evolution. Even though a genome-wide statistic summary of TEs in rye has been obtained using high-throughput DNA sequencing technology, the accurate diversity of TEs in rye, as well as their chromosomal distribution and evolution, remains elusive due to the repetitive sequence assembling problems and the high dynamic and nested nature of TEs...
2017: Frontiers in Plant Science
Katherine M Buckley, Ping Dong, R Andrew Cameron, Jonathan P Rast
Genome sequences contain all the necessary information-both coding and regulatory sequences-to construct an organism. The developmental process translates this genomic information into a three-dimensional form. One interpretation of this translation process can be described using gene regulatory network (GRN) models, which are maps of interactions among regulatory gene products in time and space. As high throughput investigations reveal increasing complexity within these GRNs, it becomes apparent that efficient methods are required to test the necessity and sufficiency of regulatory interactions...
October 17, 2017: Briefings in Functional Genomics
Weidong Huang, Yan Zhang, Yifan Chen, Yin Wang, Wensu Yuan, Ning Zhang, Toong Jin Lam, Zhiyuan Gong, Daiwen Yang, Zhi Lin
A cDNA library from a pool of all the seven silk glands from a tropical spider species was constructed. More than 1000 expressed sequence tag (EST) clones were created. Almost 65% of the EST clones were identified and around 50% were annotated. The cellular and functional distribution of the EST clones indicated high protein synthesis activity in spider silk glands. Novel clones with repetitive amino acid sequences, which is one of the most important characteristics of spider silk genes, were isolated. One of these clones, namely TuSp2 in current research, contains two almost identical fragments with one short C-terminal domain...
October 17, 2017: Scientific Reports
Takamichi Nijo, Yutaro Neriya, Hiroaki Koinuma, Nozomu Iwabuchi, Yugo Kitazawa, Kazuyuki Tanno, Yukari Okano, Kensaku Maejima, Yasuyuki Yamaji, Kenro Oshima, Shigetou Namba
Phytoplasmas are obligate intracellular parasitic bacteria that infect both plants and insects. We previously identified the sigma factor RpoD-dependent consensus promoter sequence of phytoplasma. However, the genome-wide landscape of RNA transcripts, including non-coding RNAs (ncRNAs) and RpoD-independent promoter elements, was still unknown. In this study, we performed an improved RNA sequencing analysis for genome-wide identification of the transcription start sites (TSSs) and the consensus promoter sequences...
October 17, 2017: DNA and Cell Biology
Fang-Fang Ma, Mian Wu, Ying-Na Liu, Xue-Ying Feng, Xun-Zong Wu, Jian-Qun Chen, Bin Wang
The divergence patterns of NBS - LRR genes in soybean Rsv3 locus were deciphered and several divergent alleles ( NBS_C, NBS_D and Columbia NBS_E ) were identified as the likely functional candidates of Rsv3. The soybean Rsv3 locus, which confers resistance to the soybean mosaic virus (SMV), has been previously mapped to a region containing five nucleotide binding site-leucine-rich repeats (NBS-LRR) genes (referred to as nbs_A-E) in Williams 82. In resistant cultivars, however, the number of NBS-LRR genes in this region and their divergence from susceptible alleles remain unclear...
October 16, 2017: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
Yoko Egami, Yuta Narushima, Motohiro Ohshima, Akira Yoshida, Naruki Yoneta, Yasufumi Masaki, Kunihiko Itoh
CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen)...
September 28, 2017: Journal of Biochemistry
Ahmed Allam, Michael Krauthammer
Motivation: Text and genomic data are composed of sequential tokens, such as words and nucleotides that give rise to higher order syntactic constructs. In this work, we aim at providing a comprehensive Python library implementing conditional random fields (CRFs), a class of probabilistic graphical models, for robust prediction of these constructs from sequential data. Results: Python Sequence Labeling (PySeqLab) is an open source package for performing supervised learning in structured prediction tasks...
July 21, 2017: Bioinformatics
Kun Yin, Chao Xu, Gui-Hua Zhao, Ye Liu, Ting Xiao, Song Zhu, Ge Yan
C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C351-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation...
October 16, 2017: Bioscience Trends
Tao Lin, Lihui Gao
Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector...
2018: Methods in Molecular Biology
Ruihong Zhao, Hongmei Miao, Wenqin Song, Chengbin Chen, Haiyang Zhang
Sesame (Sesamum indicum L.; family Pedaliaceae) is a commercially valuable oilseed crop with high oil content. Its small genome size favors the genomic analysis of key biological processes such as oil synthesis and metabolism. However, the 13 chromosome pairs of sesame have not been characterized because of technological limitations and their small size. To address these issues, we constructed a BAC library comprising 57,600 BAC clones for sesame. The estimated genome coverage of the sesame BAC library was 13...
October 14, 2017: Plant Biology
Muhammad Salman Haider, Cheng Zhang, Mahantesh M Kurjogi, Tariq Pervaiz, Ting Zheng, Chaobo Zhang, Chen Lide, Lingfie Shangguan, Jinggui Fang
Grapevine is an important and extensively grown fruit crop, which is severely hampered by drought worldwide. So, comprehending the impact of drought on grapevine genetic resources is necessary. In the present study, RNA-sequencing was executed using cDNA libraries constructed from both drought-stress and control plants. Results generated 12,451 differentially expressed genes (DEGs), out of which 8,021 genes were up-regulated, and 4,430 were down-regulated. Further physiological and biochemical investigations were also performed to validate the biological processes associated with the development of grapevine in response to drought stress...
October 13, 2017: Scientific Reports
Gang Chen, Jiawen Cui, Li Wang, Yingfang Zhu, Zhaogeng Lu, Biao Jin
Circular RNAs (circRNAs) are a family of transcripts with covalently closed circular structures and still largely unknown functions. Large numbers of circRNAs have been found in various biological processes in humans and animals, but fewer circRNAs have been identified in plants. We performed a genome-wide analysis of circRNAs in Arabidopsis thaliana via deep sequencing. We constructed 14 strand-specific libraries from 13 samples of plants from four developmental stages, four stress treatments, and five organs and a mixed sample across the lifespan...
2017: Frontiers in Plant Science
Jonás I Silva-Marrero, Alberto Sáez, Albert Caballero-Solares, Ivan Viegas, María Pilar Almajano, Felipe Fernández, Isabel V Baanante, Isidoro Metón
BACKGROUND: The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. RESULTS: After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp)...
October 11, 2017: BMC Genomics
Maxwell Bates, Joe Lachoff, Duncan Meech, Valentin Zulkower, Anaïs Moisy, Yisha Luo, Hille Tekotte, Cornelia Johanna Franziska Scheitz, Rupal Khilari, Florencio Mazzoldi, Deepak Chandran, Eli Groban
Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs...
October 20, 2017: ACS Synthetic Biology
Yong-Wei Yan, Bin Zou, Ting Zhu, Wael N Hozzein, Zhe-Xue Quan
RNA-seq-based SSU (small subunit) rRNA (ribosomal RNA) analysis has provided a better understanding of potentially active microbial community within environments. However, for RNA-seq library construction, high quantities of purified RNA are typically required. We propose a modified RNA-seq method for SSU rRNA-based microbial community analysis that depends on the direct ligation of a 5' adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10-100 ng) and does not require a DNA removal step...
2017: PloS One
Sean Andrew Higgins, David F Savage
A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This perspective briefly outlines the technical innovations in DNA manipulation which allow massively parallel protein biochemistry, then summarizes the methods currently available for library construction and the functional assays of protein variants...
October 9, 2017: Biochemistry
Song Yi, Ning-Ning Liu, Limei Hu, Hui Wang, Nidhi Sahni
A complete understanding of human cancer variants requires new methods to systematically and efficiently assess the functional effects of genomic mutations at a large scale. Here, we describe a set of tools to rapidly clone and stratify thousands of cancer mutations at base resolution. This protocol provides a massively parallel pipeline to achieve high stringency and throughput. The approach includes high-throughput generation of mutant clones by Gateway, confirmation of variant identity by barcoding and next-generation sequencing, and stratification of cancer variants by multiplexed interaction profiling...
November 2017: Nature Protocols
Emily Roggenkamp, Rachael M Giersch, Emily Wedeman, Muriel Eaton, Emily Turnquist, Madison N Schrock, Linah Alkotami, Thitikan Jirakittisonthon, Samantha E Schluter-Pascua, Gareth H Bayne, Cory Wasko, Megan Halloran, Gregory C Finnigan
Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome...
2017: Frontiers in Microbiology
Minho Noh, Seung Min Yoo, Won Jun Kim, Sang Yup Lee
Escherichia coli gene expression knockdown using synthetic small RNA (sRNA) can be fine-tuned by altering sRNA sequences to modulate target mRNA-binding ability, but this requires thorough checking for off-target effects. Here, we present an sRNA gene expression knockdown system fine-tuned by using different promoters to modulate synthetic sRNA abundance. Our approach entails selecting knockdown target genes resulting from in silico flux response analysis and those related to product biosynthesis then screening strains transformed with a library of synthetic sRNA-promoter combinations for enhanced production...
September 26, 2017: Cell Systems
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