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Sequence library construction

Kwang Soo Shin, Sangwoo Kim, Sung Kuk Lee
BACKGROUND: Microbial production of oleochemicals has been actively studied in the last decade. Free fatty acids (FFAs) could be converted into a variety of molecules such as industrial products, consumer products, and fuels. FFAs have been produced in metabolically engineered Escherichia coli cells expressing a signal sequence-deficient acyl-CoA thioesterase I ('TesA). Nonetheless, increasing the expression level of 'TesA seems not to be an appropriate approach to scale up FFA production because a certain ratio of each component including fatty acid synthase and 'TesA is required for optimal production of FFAs...
2016: Biotechnology for Biofuels
Zi-Xin Zhang, Shu-Niu Zhao, Gao-Feng Liu, Zu-Mei Huang, Zhen-Mu Cao, Shan-Han Cheng, Shi-Sen Lin
The Indian pepper 'Guijiangwang' (Capsicum frutescens L.), one of the world's hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper's expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000...
October 19, 2016: Scientific Reports
Zhong-Guo Shan, Ke-Xin Zhu, Fang-Yi Chen, Jie Liu, Bei Chen, Kun Qiao, Hui Peng, Ke-Jian Wang
A new gene homologous to the reported antimicrobial peptide (AMP) hyastatin from Hyas araneus was screened in the SSH library constructed from the hemocytes of Scylla paramamosain, and named SpHyastatin. In vivo study showed that SpHyastatin was predominantly expressed in hemocytes of S. paramamosain. With the challenge of either Vibrio parahaemolyticus or lipopolysaccharide (LPS), SpHyastatin showed a positive response, meaning that it was probably involved in the immune reaction against bacterial infection in vivo...
October 14, 2016: Fish & Shellfish Immunology
Fen Wei, Jie Chen, Xinye Chen, Baolong Bao
Flatfish with left-right eye asymmetry are the most significant among vertebrates. However, the genetic basis for the control of this characteristic is still unclear. We propose that the gene(s) for eye asymmetry initially control minor differences in cell number in the tissues around the eyes during development. This minor difference is then amplified, causing eye migration during metamorphosis. Therefore, comparing the neurula transcriptomes between flatfish species with different eye-reversal mutants may provide very useful information to screen for genes involved in eye asymmetry...
October 14, 2016: Gene
Amit R Majithia, Ben Tsuda, Maura Agostini, Keerthana Gnanapradeepan, Robert Rice, Gina Peloso, Kashyap A Patel, Xiaolan Zhang, Marjoleine F Broekema, Nick Patterson, Marc Duby, Ted Sharpe, Eric Kalkhoven, Evan D Rosen, Inês Barroso, Sian Ellard, Sekar Kathiresan, Stephen O'Rahilly, Krishna Chatterjee, Jose C Florez, Tarjei Mikkelsen, David B Savage, David Altshuler
Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions...
October 17, 2016: Nature Genetics
Bojiang Li, Chao Dong, Pinghua Li, Zhuqing Ren, Han Wang, Fengxiang Yu, Caibo Ning, Kaiqing Liu, Wei Wei, Ruihua Huang, Jie Chen, Wangjun Wu, Honglin Liu
Meat color is considered to be the most important indicator of meat quality, however, the molecular mechanisms underlying traits related to meat color remain mostly unknown. In this study, to elucidate the molecular basis of meat color, we constructed six cDNA libraries from biceps femoris (Bf) and soleus (Sol), which exhibit obvious differences in meat color, and analyzed the whole-transcriptome differences between Bf (white muscle) and Sol (red muscle) using high-throughput sequencing technology. Using DEseq2 method, we identified 138 differentially expressed genes (DEGs) between Bf and Sol...
October 17, 2016: Scientific Reports
Vindhya Mohindra, Ratnesh Kumar Tripathi, Akanksha Singh, Ruchi Patangia, Rajeev Kumar Singh, Kuldeep Kumar Lal, Joy Krushna Jena
The Indian catfish, Clarias magur is an air breathing fish, inhabitant of aquatic bodies characterized by low dissolved oxygen levels. It is exposed to hypoxic conditions in its natural habitat. Thus, it can be useful model to study the mechanism of hypoxia stress tolerance. In C. magur, molecular processes facilitating its adaptation to hypoxia stress remain largely unexplored, in part due to unavailability of genomic resources. The suppression subtractive hybridization technique (SSH) was employed to compare the differential expression of transcripts under experimental hypoxic conditions, to that of normoxic conditions...
October 11, 2016: Fish & Shellfish Immunology
Xiaojie Yang, Yuanming Zhao, Deyi Xie, Yao Sun, Xunlu Zhu, Nardana Esmaeili, Zuoren Yang, Ye Wang, Guo Yin, Shuping Lv, Lihong Nie, Zhongjie Tang, Fu'an Zhao, Wu Li, Neelam Mishra, Li Sun, Wei Zhu, Weiping Fang
Hybrid vigor contributes in a large way to the yield and quality of cotton (Gossypium hirsutum) fiber. Although microRNAs play essential regulatory roles in flower induction and development, it is still unclear if microRNAs are involved in male sterility, as the regulatory molecular mechanisms of male sterility in cotton need to be better defined. In this study, two independent small RNA libraries were constructed and sequenced from the young buds collected from the sporogenous cell formation to the meiosis stage of the male sterile line Yu98-8A and the near-isogenic line...
October 7, 2016: International Journal of Molecular Sciences
Shreyas Shah, Kiran Jadhav, Vandana Shah, Nidhi Gupta, Kapil Dagrus
: The vast majority of cancer epigenetic research is now focused on micro RNA (miRNA). Though thousands of miRNA have been identified, the validation of their role is a continuous process. AIM: the aim of this paper is to comprehensively review the role of miRNA 21 in Oral cancer as a marker for diagnosis, prognosis and therapeutic target. METHOD: The data was collected from major search engines like PubMed, Science Direct, Cochrane library and Google Patents with the key words miRNA 21, miRNA and Oral Cancer, miRNA 21 prognostic role, miRNA therapeutic target etc...
September 19, 2016: MicroRNA
Letícia Maria Menezes Nóbrega, Francisco Montagner, Adriana Costa Ribeiro, Márcia Alves Pinto Mayer, Brenda Paula Figueiredo de Almeida Gomes
The aim of this study was to explore the bacterial diversity of 10 root canals with acute apical abscess using clonal analysis. Samples were collected from 10 patients and submitted to bacterial DNA isolation, 16S rRNA gene amplification, cloning, and sequencing. A bacterial genomic library was constructed and bacterial diversity was estimated. The mean number of taxa per canal was 15, ranging from 11 to 21. A total of 689 clones were analyzed and 76 phylotypes identified, of which 47 (61.84%) were different species and 29 (38...
October 10, 2016: Brazilian Oral Research
I R Sasselli, C G Pappas, E Matthews, T Wang, N T Hunt, R V Ulijn, T Tuttle
Despite progress, a fundamental understanding of the relationships between the molecular structure and self-assembly configuration of Fmoc-dipeptides is still in its infancy. In this work, we provide a combined experimental and computational approach that makes use of free energy equilibration of a number of related Fmoc-dipeptides to arrive at an atomistic model of Fmoc-threonine-phenylalanine-amide (Fmoc-TF-NH2) which forms twisted fibres. By using dynamic peptide libraries where closely related dipeptide sequences are dynamically exchanged to eventually favour the formation of the thermodynamically most stable configuration, the relative importance of C-terminus modifications (amide versus methyl ester) and contributions of aliphatic versus aromatic amino acids (phenylalanine F vs...
October 12, 2016: Soft Matter
Weiyan Gong, Yong Liu, Zhihong Wu, Shaohai Wang, Guixing Qiu, Shouqing Lin
The present study was performed to identify and characterize genes involved in osteoblasts function. Firstly, we constructed and sequenced a human osteoblast full-length cDNA library to screen for genes whose functions have not been reported and further identify these candidate genes through detecting the relationship with the activator protein-1 (AP-1) transcription factor complex using a dual luciferase reporter system. Only one gene, namely METRNL (Meteorin, glial cell differentiation regulator-like) has been screened out...
2016: PloS One
Libao Cheng, Shuyan Li, Sainan Chen, Yan Wang, Meizhen Yu, Xuehao Chen, Liangjun Li, Jingjing Yin
The product organ (storage organ; corm) of the Chinese water chestnut has become a very popular food in Asian countries because of its unique nutritional value. Corm formation is a complex biological process, and extensive whole genome analysis of transcripts during corm development has not been carried out. In this study, four corm libraries at different developmental stages were constructed, and gene expression was identified using a high-throughput tag sequencing technique. Approximately 4.9 million tags were sequenced, and 4,371,386, 4,372,602, 4,782,494, and 5,276,540 clean tags, including 119,676, 110,701, 100,089, and 101,239 distinct tags, respectively, were obtained after removal of low-quality tags from each library...
2016: PloS One
Weilan Shao, Kesen Ma, Yilin Le, Hongcheng Wang, Chong Sha
Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution...
2017: Methods in Molecular Biology
Alicia M Jones, Joshua T Atkinson, Jonathan J Silberg
Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein...
2017: Methods in Molecular Biology
G S C Buso, A M M Reis, Z P S Amaral, M E Ferreira
This study was undertaken primarily to develop new simple sequence repeat (SSR) markers for Capsicum. As part of this project aimed at broadening the use of molecular tools in Capsicum breeding, two genomic libraries enriched for AG/TC repeat sequences were constructed for Capsicum annuum. A total of 475 DNA clones were sequenced from both libraries and 144 SSR markers were tested on cultivated and wild species of Capsicum. Forty-five SSR markers were randomly selected to genotype a panel of 48 accessions of the Capsicum germplasm bank...
September 23, 2016: Genetics and Molecular Research: GMR
Linsong Dong, Shijun Xiao, Junwei Chen, Liang Wan, Zhiyong Wang
Genomic selection (GS) is an effective method to improve predictive accuracies of genetic values. However, high cost in genotyping will limit the application of this technology in some species. Therefore, it is necessary to find some methods to reduce the genotyping costs in genomic selection. Large yellow croaker is one of the most commercially important marine fish species in southeast China and Eastern Asia. In this study, genotyping-by-sequencing was used to construct the libraries for the NGS sequencing and find 29,748 SNPs in the genome...
October 4, 2016: Marine Biotechnology
Bin Song, Juan Du, Ye Feng, Yong-Jian Gao, Ji-Sheng Zhao
The aim of the present study was to investigate the mechanisms of long non-coding RNAs (lncRNAs) in a gastric cancer cell line treated with celecoxib. The human gastric carcinoma cell line NCI-N87 was treated with 15 µM celecoxib for 72 h (celecoxib group) and an equal volume of dimethylsulfoxide (control group), respectively. Libraries were constructed by NEBNext Ultra RNA Library Prep kit for Illumina. Paired-end RNA sequencing reads were aligned to a human hg19 reference genome using TopHat2. Differentially expressed genes (DEGs) and lncRNAs were identified using Cuffdiff...
October 2016: Experimental and Therapeutic Medicine
Fanggui Zhao, Chen Wang, Jian Han, Xudong Zhu, Xiaopeng Li, Xicheng Wang, Jinggui Fang
MicroRNAs (miRNAs) are critical regulators of various biological and metabolic processes of plants. Numerous miRNAs and their functions have been identified and analyzed in many plants. However, till now, the involvement of miRNAs in the response of grapevine berries to ethylene has not been reported yet. Here, Solexa technology was employed to deeply sequence small RNA libraries constructed from grapevine berries treated with and without ethylene. A total of 124 known and 78 novel miRNAs were identified. Among these miRNAs, 162 miRNAs were clearly responsive to ethylene, with 55 downregulated, 59 upregulated, and 14 unchanged miRNAs detected only in the control...
September 30, 2016: Functional & Integrative Genomics
Gabe Haller, David Alvarado, Kevin McCall, Robi D Mitra, Matthew B Dobbs, Christina A Gurnett
Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation...
October 3, 2016: Nature Methods
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