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High-Throughput Sequencing

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https://www.readbyqxmd.com/read/28103239/identifying-t-cell-receptors-from-high-throughput-sequencing-dealing-with-promiscuity-in-tcr%C3%AE-and-tcr%C3%AE-pairing
#1
Edward S Lee, Paul G Thomas, Jeff E Mold, Andrew J Yates
Characterisation of the T cell receptors (TCR) involved in immune responses is important for the design of vaccines and immunotherapies for cancer and autoimmune disease. The specificity of the interaction between the TCR heterodimer and its peptide-MHC ligand derives largely from the juxtaposed hypervariable CDR3 regions on the TCRα and TCRβ chains, and obtaining the paired sequences of these regions is a standard for functionally defining the TCR. A brute force approach to identifying the TCRs in a population of T cells is to use high-throughput single-cell sequencing, but currently this process remains costly and risks missing small clones...
January 19, 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28103009/fully-automated-one-step-synthesis-of-single-transcript-talen-pairs-using-a-biological-foundry
#2
Ran Chao, Jing Liang, Ipek Tasan, Tong Si, Linyang Ju, Huimin Zhao
Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present an assembly scheme to synthesize and express a pair of TALENs in a single transcript format with the help of a P2A self-cleavage sequence...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28103008/quantitative-tracking-of-combinatorially-engineered-populations-with-multiplexed-binary-assemblies
#3
Ramsey Zeitoun, Gur Pines, William C Grau, Ryan T Gill
Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory timescales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28102441/the-effect-of-land-use-on-bacterial-communities-in-saline-alkali-soil
#4
Mu Peng, Hongbai Jia, Qiuyu Wang
Saline-alkali soil can inhibit the growth of crops as a consequence of cellular damage through oxidation of lipids and proteins and degradation of nucleic acids, ultimately leading to cell death. The bacterial community composition and diversity in saline-alkali soil across different land uses, such as agricultural land, forest land, and grassland, were evaluated using high-throughput sequencing of the bacterial 16S rRNA gene. Significant differences in the soil physicochemical characteristics and bacterial community among different land uses were observed in this study...
January 19, 2017: Current Microbiology
https://www.readbyqxmd.com/read/28100695/mapping-the-affinity-landscape-of-thrombin-binding-aptamers-on-2-f-ana-dna-chimeric-g-quadruplex-microarrays
#5
Jory Lietard, Hala Abou Assi, Irene Gómez-Pinto, Carlos González, Mark M Somoza, Masad J Damha
In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered...
January 18, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28100584/umi-tools-modelling-sequencing-errors-in-unique-molecular-identifiers-to-improve-quantification-accuracy
#6
Tom Sean Smith, Andreas Heger, Ian Sudbery
Unique Molecular Identifiers (UMIs) are random oligonucleotide barcodes that are increasingly used in high-throughput sequencing experiments. Through a UMI, identical copies arising from distinct molecules can be distinguished from those arising through PCR-amplification of the same molecule. However, bioinformatic methods to leverage the information from UMIs have yet to be formalised. In particular, sequencing errors in the UMI sequence are often ignored, or else resolved in an ad-hoc manner. We show that errors in the UMI sequence are common and introduce network based methods to account for these errors when identifying PCR duplicates...
January 18, 2017: Genome Research
https://www.readbyqxmd.com/read/28100302/fecal-virome-of-healthy-chickens-reveals-a-large-diversity-of-the-eukaryote-viral-community-including-novel-circular-single-stranded-dna-viruses
#7
Diane Lima, Samuel Paulo Cibulski, Fabrine Finkler, Thais Teixeira, Ana Paula Varela, Cristine Cerva, Márcia Loiko, Camila Scheffer, Helton Dos Santos, Fabiana Mayer, Paulo Roehe
This study is focused on the identification of the fecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7,743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with BLASTx revealed that 279 contigs (4%) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes...
January 18, 2017: Journal of General Virology
https://www.readbyqxmd.com/read/28100189/an-snp-based-saturated-genetic-map-and-qtl-analysis-of-fruit-related-traits-in-zucchini-using-genotyping-by-sequencing
#8
Javier Montero-Pau, José Blanca, Cristina Esteras, Eva Ma Martínez-Pérez, Pedro Gómez, Antonio J Monforte, Joaquín Cañizares, Belén Picó
BACKGROUND: Cucurbita pepo is a cucurbit with growing economic importance worldwide. Zucchini morphotype is the most important within this highly variable species. Recently, transcriptome and Simple Sequence Repeat (SSR)- and Single Nucleotide Polymorphism (SNP)-based medium density maps have been reported, however further genomic tools are needed for efficient molecular breeding in the species. Our objective is to combine currently available complete transcriptomes and the Zucchini genome sequence with high throughput genotyping methods, mapping population development and extensive phenotyping to facilitate the advance of genomic research in this species...
January 18, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28100185/an-improved-genome-assembly-uncovers-prolific-tandem-repeats-in-atlantic-cod
#9
Ole K Tørresen, Bastiaan Star, Sissel Jentoft, William B Reinar, Harald Grove, Jason R Miller, Brian P Walenz, James Knight, Jenny M Ekholm, Paul Peluso, Rolf B Edvardsen, Ave Tooming-Klunderud, Morten Skage, Sigbjørn Lien, Kjetill S Jakobsen, Alexander J Nederbragt
BACKGROUND: The first Atlantic cod (Gadus morhua) genome assembly published in 2011 was one of the early genome assemblies exclusively based on high-throughput 454 pyrosequencing. Since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. The development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies...
January 18, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28100169/medium-throughput-bisulfite-sequencing-for-accurate-detection-of-5-methylcytosine-and-5-hydroxymethylcytosine
#10
Gary G Chen, Jeffrey A Gross, Pierre-Eric Lutz, Kathryn Vaillancourt, Gilles Maussion, Alexandre Bramoulle, Jean-François Théroux, Elena S Gardini, Ulrike Ehlert, Geneviève Bourret, Aurélie Masurel, Pierre Lepage, Naguib Mechawar, Gustavo Turecki, Carl Ernst
BACKGROUND: Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing. RESULTS: Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution...
January 18, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28099913/zibupiyin-recipe-improves-cognitive-decline-by-regulating-gut-microbiota-in-zucker-diabetic-fatty-rats
#11
Chunyan Gu, Wen Zhou, Wang Wang, Hong Xiang, Huiying Xu, Lina Liang, Hua Sui, Libin Zhan, Xiaoguang Lu
Numerous researches supported that microbiota can influence behavior and modulate cognitive function through "microbiota-gut-brain" axis. Our previous study has demonstrated that ZiBuPiYin recipe (ZBPYR) possesses excellent pharmacological effects against diabetes-associated cognitive decline. To elucidate the role of ZBPYR in regulating the balance of gut microbiota to improve psychological-stress-induced diabetes-associated cognitive decline (PSDACD), we compared blood glucose, behavioral and cognitive functions and diversity of the bacterial community among experimental groups...
January 12, 2017: Oncotarget
https://www.readbyqxmd.com/read/28099906/genomic-characteristics-of-pancreatic-squamous-cell-carcinoma-an-investigation-by-using-high-throughput-sequencing-after-in-solution-hybrid-capture
#12
Meng-Dan Xu, Shu-Ling Liu, Yi-Zhong Feng, Qiang Liu, Meng Shen, Qiaoming Zhi, Zeyi Liu, Dong-Mei Gu, Jie Yu, Liu-Mei Shou, Fei-Ran Gong, Qi Zhu, Weiming Duan, Kai Chen, Junning Zhang, Meng-Yao Wu, Min Tao, Wei Li
Squamous cell carcinoma (SCC) of pancreas is a rare histotype of pancreatic ductal carcinoma which is distinct from pancreatic adenocarcinoma (AC). Although there are standard treatments for pancreatic AC, no precise therapies exist for pancreatic SCC. Here, we screened 1033 cases of pancreatic cancer and identified 2 cases of pure SCC, which were pathologically diagnosed on the basis of finding definite intercellular bridges and/or focal keratin peal formation in the tumor cells. Immunohistochemistry assay confirmed the positive expression of CK5/6 and p63 in pancreatic SCC...
January 16, 2017: Oncotarget
https://www.readbyqxmd.com/read/28099455/evaluating-the-impact-of-dna-extraction-method-on-the-representation-of-human-oral-bacterial-and-fungal-communities
#13
Anna Vesty, Kristi Biswas, Michael W Taylor, Kim Gear, Richard G Douglas
The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3-V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi...
2017: PloS One
https://www.readbyqxmd.com/read/28099430/pooled-crispr-screening-with-single-cell-transcriptome-readout
#14
Paul Datlinger, André F Rendeiro, Christian Schmidl, Thomas Krausgruber, Peter Traxler, Johanna Klughammer, Linda C Schuster, Amelie Kuchler, Donat Alpar, Christoph Bock
CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells...
January 18, 2017: Nature Methods
https://www.readbyqxmd.com/read/28098358/subclinical-alteration-of-the-cervical-vaginal-microbiome-in-women-with-idiopathic-infertility
#15
Giuseppina Campisciano, Fiorella Florian, Angela D'Eustacchio, David Stanković, Giuseppe Ricci, Manola Comar
Biomarkers have a wide application in research and clinic, they help to choose the correct treatment for diseases. Recent studies, addressing the vaginal microbiome using next generation sequencing (NGS), reported the involvement of bacterial species in infertility. We compared the vaginal microbiome of idiopathic infertility diagnosed women with that of healthy, non-idiopathic infertile and bacterial vaginosis affected women, to identify bacterial species suitable as biomarkers. Information on microorganisms was obtained from the V3-16S rDNA sequencing of cervical-vaginal fluids of 96 women using the Ion Torrent platform...
January 18, 2017: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/28097059/the-complete-chloroplast-genome-sequence-of-an-endemic-monotypic-genus-hagenia-rosaceae-structural-comparative-analysis-gene-content-and-microsatellite-detection
#16
Andrew W Gichira, Zhizhong Li, Josphat K Saina, Zhicheng Long, Guangwan Hu, Robert W Gituru, Qingfeng Wang, Jinming Chen
Hagenia is an endangered monotypic genus endemic to the topical mountains of Africa. The only species, Hagenia abyssinica (Bruce) J.F. Gmel, is an important medicinal plant producing bioactive compounds that have been traditionally used by African communities as a remedy for gastrointestinal ailments in both humans and animals. Complete chloroplast genomes have been applied in resolving phylogenetic relationships within plant families. We employed high-throughput sequencing technologies to determine the complete chloroplast genome sequence of H...
2017: PeerJ
https://www.readbyqxmd.com/read/28097056/rhea-a-transparent-and-modular-r-pipeline-for-microbial-profiling-based-on-16s-rrna-gene-amplicons
#17
Ilias Lagkouvardos, Sandra Fischer, Neeraj Kumar, Thomas Clavel
The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs...
2017: PeerJ
https://www.readbyqxmd.com/read/28097054/hydrogel-scaffolds-promote-neural-gene-expression-and-structural-reorganization-in-human-astrocyte-cultures
#18
V Bleu Knight, Elba E Serrano
Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene...
2017: PeerJ
https://www.readbyqxmd.com/read/28096217/high-throughput-analysis-reveals-novel-maternal-germline-rnas-crucial-for-primordial-germ-cell-preservation-and-proper-migration
#19
Dawn A Owens, Amanda M Butler, Tristan H Aguero, Karen M Newman, Derek Van Booven, Mary Lou King
During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA-sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively...
January 15, 2017: Development
https://www.readbyqxmd.com/read/28096084/timesvector-a-vectorized-clustering-approach-to-the-analysis-of-time-series-transcriptome-data-from-multiple-phenotypes
#20
Inuk Jung, Kyuri Jo, Hyejin Kang, Hongryul Ahn, Youngjae Yu, Sun Kim
MOTIVATION: Identifying biologically meaningful gene expression patterns from time series gene expression data is important to understand the underlying biological mechanisms. To identify significantly perturbed gene sets between different phenotypes, analysis of time series transcriptome data requires consideration of time and sample dimensions. Thus, the analysis of such time series data seeks to search gene sets that exhibit similar or different expression patterns between two or more sample conditions, constituting the three-dimensional data, i...
January 17, 2017: Bioinformatics
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