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Sequence library construction

Rene Geissler, Andrew Grimson
GFP reporter constructs are widely used as an expression system for studying the function of regulatory sequence motifs (cis elements) within the 3'-UTRs (3' untranslated regions) of mRNAs. Here we provide details on the characterization of individual sequence motifs, which typically regulate mRNA decay and translation. In addition, we describe methods to identify trans factors required for the function of such elements. To facilitate efficient identification of novel functional 3'-UTR motifs, we describe a screening approach based on dual-color fluorescence reporter constructs...
2018: Methods in Molecular Biology
Shujun Ou, Ning Jiang
Long terminal-repeat retrotransposons (LTR-RTs) are prevalent in plant genomes. Identification of LTR-RTs is critical for achieving high-quality gene annotation. Based on the well-conserved structure, multiple programs were developed for de novo identification of LTR-RTs; however, these programs are associated with low specificity and high false discovery rate (FDR). Here we report LTR_retriever, a multithreading empowered Perl program that identifies LTR-RTs and generates high-quality LTR libraries from genomic sequences...
December 12, 2017: Plant Physiology
Steven J M Jones, Gregory A Taylor, Simon Chan, René L Warren, S Austin Hammond, Steven Bilobram, Gideon Mordecai, Curtis A Suttle, Kristina M Miller, Angela Schulze, Amy M Chan, Samantha J Jones, Kane Tse, Irene Li, Dorothy Cheung, Karen L Mungall, Caleb Choo, Adrian Ally, Noreen Dhalla, Angela K Y Tam, Armelle Troussard, Heather Kirk, Pawan Pandoh, Daniel Paulino, Robin J N Coope, Andrew J Mungall, Richard Moore, Yongjun Zhao, Inanc Birol, Yussanne Ma, Marco Marra, Martin Haulena
The beluga whale is a cetacean that inhabits arctic and subarctic regions, and is the only living member of the genus Delphinapterus. The genome of the beluga whale was determined using DNA sequencing approaches that employed both microfluidic partitioning library and non-partitioned library construction. The former allowed for the construction of a highly contiguous assembly with a scaffold N50 length of over 19 Mbp and total reconstruction of 2.32 Gbp. To aid our understanding of the functional elements, transcriptome data was also derived from brain, duodenum, heart, lung, spleen, and liver tissue...
December 11, 2017: Genes
Samantha J Jones, Martin Haulena, Gregory A Taylor, Simon Chan, Steven Bilobram, René L Warren, S Austin Hammond, Karen L Mungall, Caleb Choo, Heather Kirk, Pawan Pandoh, Adrian Ally, Noreen Dhalla, Angela K Y Tam, Armelle Troussard, Daniel Paulino, Robin J N Coope, Andrew J Mungall, Richard Moore, Yongjun Zhao, Inanc Birol, Yussanne Ma, Marco Marra, Steven J M Jones
The northern sea otter inhabits coastal waters of the northern Pacific Ocean and is the largest member of the Mustelidae family. DNA sequencing methods that utilize microfluidic partitioned and non-partitioned library construction were used to establish the sea otter genome. The final assembly provided 2.426 Gbp of highly contiguous assembled genomic sequences with a scaffold N50 length of over 38 Mbp. We generated transcriptome data derived from a lymphoma to aid in the determination of functional elements...
December 11, 2017: Genes
Jing Zhang, Yanlin Song, Min He, Qingqing Ren, Yunhui Zeng, Zhiyong Liu, Hao Liu, Jianguo Xu
OBJECT: The aim of this study was to systematically evaluate the diagnostic performance of increased signal intensity within the resection cavity on fluid-attenuated inversion recovery (FLAIR) sequences for detection of progression in patients with glioma through performing a meta-analysis. METHODS: PubMed, Web of Science, EMBASE, Cochrane Library and China National Knowledge Infrastructure were searched for potentially relevant literature. The study characteristics and relevant data were extracted...
December 8, 2017: World Neurosurgery
M Reyes Vidal-Acuña, Maite Ruiz-Pérez de Pipaón, María José Torres-Sánchez, Javier Aznar
An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS...
December 8, 2017: Medical Mycology: Official Publication of the International Society for Human and Animal Mycology
Jing Leng, Xuchuan Liu, Chunyong Zhang, Renjun Zhu, Huaming Mao
A total of 6,219 positive clones were obtained by constructing a BAC library of uncultured ruminal fungi of gayal, and two clones (xynF1 and eglF2) with lignocellulolytic enzyme activity were selected. The sequencing results showed that xynF1 and eglF2 had 903-bp, and 1,995-bp, open reading frames likely to encode β-xylanase (XynF1) and β-glucosidase (EglF2), respectively. The amino acid sequence of XynF1 had 99% coverage and 95% homology to the endo-β-1,4-xylanase encoded by the cellulase gene of Orpinomyces sp...
December 8, 2017: Journal of General and Applied Microbiology
Fumihito Miura, Takashi Ito
Post-bisulfite adaptor tagging (PBAT) is a highly efficient procedure to construct libraries for whole-genome bisulfite sequencing (WGBS). PBAT attaches adaptors to bisulfite-converted genomic DNA to circumvent bisulfite-induced degradation of library DNA inherent to conventional WGBS protocols. Consequently, it enables PCR-free WGBS from nanogram quantities of mammalian DNA, thereby serving as an invaluable tool for methylomics.
2018: Methods in Molecular Biology
Roland Prielhofer, Juan J Barrero, Stefanie Steuer, Thomas Gassler, Richard Zahrl, Kristin Baumann, Michael Sauer, Diethard Mattanovich, Brigitte Gasser, Hans Marx
BACKGROUND: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P...
December 8, 2017: BMC Systems Biology
Y Cao, K N Zou, J P Huang, K Ma, Y Ping
OBJECTIVES: To analyze and detect the whole genome sequence of human mitochondrial DNA (mtDNA) by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. METHODS: Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer...
August 2017: Fa Yi Xue za Zhi
Ying Lan, Yanwu Li, Zhiguo E, Feng Sun, Linlin Du, Qiufang Xu, Tong Zhou, Yijun Zhou, Yongjian Fan
RNA interference (RNAi) is a conserved mechanism against viruses in plants and animals. It is thought to inactivate the viral genome by producing virus-derived small interfering RNAs (vsiRNAs). Rice black-streaked dwarf virus (RBSDV) is transmitted to plants by the small brown planthopper (Laodelphax striatellus), and seriously threatens production of rice in East Asia, particularly Oryza sativa japonica subspecies. Through deep sequencing, genome-wide comparisons of RBSDV-derived vsiRNAs were made between the japonica variety Nipponbare, and the indica variety 9311...
December 7, 2017: Journal of Basic Microbiology
April S Gislason, Keith Turner, Mike Domaratzki, Silvia T Cardona
Burkholderia cenocepacia K56-2 belongs to the Burkholderia cepacia complex, a group of Gram-negative opportunistic pathogens that have large and dynamic genomes. In this work, we identified the essential genome of B. cenocepacia K56-2 using high-density transposon mutagenesis and insertion site sequencing (Tn-seq circle). We constructed a library of one million transposon mutants and identified the transposon insertions at an average of one insertion per 27 bp. The probability of gene essentiality was determined by comparing of the insertion density per gene with the variance of neutral datasets generated by Monte Carlo simulations...
November 2017: Microbial Genomics
Zujun Yin, Xiulan Han, Yan Li, Junjuan Wang, Delong Wang, Shuai Wang, Xiaoqiong Fu, Wuwei Ye
Small RNAs play an important role in regulating plant responses to abiotic stress. Depending on the method of salt application, whether sudden or gradual, plants may experience either salt shock or salt stress, respectively. In this study, small RNA expression in response to salt shock and long-term salt stress in parallel experiments was described. Cotton small RNA libraries were constructed and sequenced under normal conditions, as well as sudden and gradual salt application. A total of 225 cotton microRNAs (miRNAs) were identified and of these 24 were novel miRNAs...
December 5, 2017: Genes
Lidan Ye, Chengcheng Yang, Hongwei Yu
With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes...
November 27, 2017: Applied Microbiology and Biotechnology
Christopher Wai Siong Ng, Bustamin Kosmo, Peak-Ling Lee, Chun Kiat Lee, Jingxue Guo, Zhaojin Chen, Lily Chiu, Hong Kai Lee, Sherry Ho, Jianbiao Zhou, Mingxuan Lin, Karen M L Tan, Kenneth H K Ban, Tin Wee Tan, Wee Joo Chng, Benedict Yan
AIM: The presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated...
November 27, 2017: Journal of Clinical Pathology
Kishor Dhaygude, Kalevi Trontti, Jenni Paviala, Claire Morandin, Christopher Wheat, Liselotte Sundström, Heikki Helanterä
Transcriptome resources for social insects have the potential to provide new insight into polyphenism, i.e., how divergent phenotypes arise from the same genome. Here we present a transcriptome based on paired-end RNA sequencing data for the ant Formica exsecta (Formicidae, Hymenoptera). The RNA sequencing libraries were constructed from samples of several life stages of both sexes and female castes of queens and workers, in order to maximize representation of expressed genes. We first compare the performance of common assembly and scaffolding software (Trinity, Velvet-Oases, and SOAPdenovo-trans), in producing de novo assemblies...
2017: PeerJ
Mária Bučková, Andrea Puškárová, Katarína Ženišová, Lucia Kraková, Ľubica Piknová, Tomáš Kuchta, Domenico Pangallo
Culture-dependent and culture-independent strategies were applied to investigate the microbiota of autumn undamaged and damaged berries, winter berries and ice wine must samples of Grüner Veltliner (Veltlínske zelené) from Small Carpathian wine-producing region. One hundred twenty-six yeasts and 242 bacterial strains isolated from several microbiological media (YPD, PDA, R2A, GYC, MRS and MRS-T) were clustered by ITS-PCR and subsequent Qiaxcel electrophoresis. Representatives of each cluster were identified by sequencing...
November 15, 2017: International Journal of Food Microbiology
Manfred T Reetz, Aitao Li, Carlos G Acevedo-Rocha, Zhoutong Sun, Tony Cox, Jia Lucy Xu
Saturation mutagenesis (SM) constitutes a widely used technique in directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Here we show how this fundamental problem can be solved by applying high-fidelity solid-phase chemical gene synthesis on Silicon-chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with stereoselective formation of (R,R)- and (S,S)-cyclohexane-1,2-diol...
November 24, 2017: Chembiochem: a European Journal of Chemical Biology
Sean M Halper, Daniel P Cetnar, Howard M Salis
Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels...
2018: Methods in Molecular Biology
Jingliang Kang, Xiuhui Ma, Shunping He
BACKGROUND: Organisms living at high altitudes face low oxygen and temperature conditions; thus, the genetic mechanisms underlying the adaptations in these organisms merit investigation. The glyptosternoid fish, Creteuchiloglanis macropterus mainly inhabits regions with gradual increases in altitudes along the Nujiang River and might serve as an appropriate evolutionary model for detecting adaptation processes in environments with altitude changes. RESULTS: We constructed eleven RNA-sequencing (RNA-seq) libraries of C...
November 23, 2017: BMC Evolutionary Biology
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