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Alexander B Rosenberg, Charles M Roco, Richard A Muscat, Anna Kuchina, Paul Sample, Zizhen Yao, Lucas Gray, David J Peeler, Sumit Mukherjee, Wei Chen, Suzie H Pun, Drew L Sellers, Bosiljka Tasic, Georg Seelig
To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. Over 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation...
March 15, 2018: Science
He Chen, Jiacheng Yao, Yusi Fu, Yuhong Pang, Jianbin Wang, Yanyi Huang
The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically labeling adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing...
March 15, 2018: ACS Applied Materials & Interfaces
Linkun Wu, Jun Chen, Zhigang Xiao, Xiaocheng Zhu, Juanying Wang, Hongmiao Wu, Yanhong Wu, Zhongyi Zhang, Wenxiong Lin
The production and quality of Rehmannia glutinosa can be dramatically reduced by replant disease under consecutive monoculture. The root-associated microbiome, also known as the second genome of the plant, was investigated to understand its impact on plant health. Culture-dependent and culture-independent pyrosequencing analysis was applied to assess the shifts in soil bacterial communities in the rhizosphere and rhizoplane under consecutive monoculture. The results show that the root-associated microbiome (including rhizosphere and rhizoplane microbiomes) was significantly impacted by rhizocompartments and consecutive monoculture...
March 14, 2018: International Journal of Molecular Sciences
Beiyuan Fan, Junbo Wang, Ying Xu, Jian Chen
Quantification of single-cell proteomics provides key insights in the field of cellular heterogeneity. This chapter discusses the emerging techniques that are being used to measure the protein copy numbers at the single-cell level, which includes flow cytometry, mass cytometry, droplet cytometry, microengraving, and single-cell barcoding microchip. The advantages and limitations of each technique are compared, and future research opportunities are highlighted.
2018: Methods in Molecular Biology
Seynabou Séne, Marc-André Selosse, Mathieu Forget, Josie Lambourdière, Khoudia Cissé, Abdala Gamby Diédhiou, Elsie Rivera-Ocasio, Hippolyte Kodja, Norikazu Kameyama, Kazuhide Nara, Lucie Vincenot, Jean-Louis Mansot, Jean Weber, Mélanie Roy, Samba Ndao Sylla, Amadou Bâ
Global trade increases plant introductions, but joint introduction of associated microbes is overlooked. We analyzed the ectomycorrhizal fungi of a Caribbean beach tree, seagrape (Coccoloba uvifera, Polygonacaeae), introduced pantropically to stabilize coastal soils and produce edible fruits. Seagrape displays a limited symbiont diversity in the Caribbean. In five regions of introduction (Brazil, Japan, Malaysia, Réunion and Senegal), molecular barcoding showed that seagrape mostly or exclusively associates with Scleroderma species (Basidiomycota) that were hitherto only known from Caribbean seagrape stands...
March 13, 2018: ISME Journal
M Summa, H Henttonen, L Maunula
Human noroviruses (HuNoVs) are one of the leading global causes of diarrhoeal diseases and are transmitted mainly from person to person but also through contaminated food, water and fomites. The possible zoonotic nature of NoVs has occasionally been discussed, although the viruses are generally considered to be host-species-specific. We investigated whether wild birds and rodents could serve as carriers of HuNoVs, thereby transmitting the virus to humans directly or indirectly by contaminating foods. All samples, 115 avian and 100 rat faeces collected in springs 2009-2013 from dump sites, and 85 faeces from yellow-necked mice trapped in late autumn 2008 and 2009 after the rodents entered human settlements due to the first night frosts, were screened for HuNoV using real-time reverse transcription PCR...
March 12, 2018: Zoonoses and Public Health
Manuel Elías-Gutiérrez, Martha Valdez-Moreno, Janet Topan, Monica R Young, José Angel Cohuo-Colli
Currently, freshwater zooplankton sampling and identification methodologies have remained virtually unchanged since they were first established in the beginning of the XX century. One major contributing factor to this slow progress is the limited success of modern genetic methodologies, such as DNA barcoding, in several of the main groups. This study demonstrates improved protocols which enable the rapid assessment of most animal taxa inhabiting any freshwater system by combining the use of light traps, careful fixation at low temperatures using ethanol, and zooplankton-specific primers...
March 2018: Ecology and Evolution
Jinho Lee, Gary K Geiss, Gokhan Demirkan, Christopher P Vellano, Brian Filanoski, Yiling Lu, Zhenlin Ju, Shuangxing Yu, Huifang Guo, Lisa Y Bogatzki, Warren E Carter, Rhonda K Meredith, Savitri Krishnamurthy, Zhiyong Ding, Joseph M Beechem, Gordon Mills
Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy.  Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application.  Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples...
March 12, 2018: Molecular & Cellular Proteomics: MCP
Steven Parker, Marcin G Fraczek, Jian Wu, Sara Shamsah, Alkisti Manousaki, Kobchai Dungrattanalert, Rogerio Alves de Almeida, Edith Invernizzi, Tim Burgis, Walid Omara, Sam Griffiths-Jones, Daniela Delneri, Raymond T O'Keefe
Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unannotated transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media...
March 12, 2018: PLoS Genetics
Cloe S Pogoda, Kyle G Keepers, Sarah E Hamsher, Joshua G Stepanek, Nolan C Kane, J Patrick Kociolek
Diatoms are the most diverse lineage of algae and at the base of most aquatic food webs, but only 11 of their mitochondrial genomes have been described. Herein, we present the mitochondrial genomes of six diatom species, including: Melosira undulata, Nitzschia alba, Surirella sp., Entomoneis sp., Halamphora coffeaeformis, and Halamphora calidilacuna. Comparison of these six genomes to the 11 currently published diatom mitochondrial genomes revealed a novel ubiquitous feature block consisting of tatC-orf157-rps11...
March 11, 2018: Mitochondrial DNA. Part A. DNA Mapping, Sequencing, and Analysis
Satisha Hegde, Archana Saini, Harsha Vasudev Hegde, Sanjiva D Kholkute, Subarna Roy
Saraca asoca (Roxb.) De Wilde is an important medicinal plant from the Western Ghats of India, traditionally used in treatment of various gynecological disorders. Increasing commercial demand and decreasing numbers has resulted in this plant becoming endangered with crude drug materials being extensively substituted/adulterated with other plant species. The present study was undertaken with the objective of development and evaluation of multivariate cluster analysis of ISSR fingerprints against rbcL -based DNA barcodes as tool to understand the relationships and to differentiate common adulterants and substituents from S...
March 2018: 3 Biotech
Pavel Bashtrykov, Albert Jeltsch
Methylation of cytosine bases in DNA is one of the main epigenetic signals regulating gene expression and chromatin structure. The distribution of DNA methylation in the genome has a cell-type-specific pattern and can be modulated by internal or external stimuli. One of the most powerful approaches to investigate DNA methylation patterns is bisulfite conversion of the DNA followed by DNA sequencing, which allows to determine methylation patterns at a single-cytosine resolution. Here, we present a protocol for bisulfite DNA methylation analysis of targeted genomic regions using amplicon-based next-generation sequencing (NGS) on an Illumina sequencing system...
2018: Methods in Molecular Biology
Birthe Thormann, Dirk Ahrens, Carlos Iván Espinosa, Diego Marín Armijos, Thomas Wagner, Johann W Wägele, Marcell K Peters
Elevational diversity gradients are typically studied without considering the complex small-scale topography of large mountains, which generates habitats of strongly different environmental conditions within the same elevational zones. Here we analyzed the importance of small-scale topography for elevational diversity patterns of hyperdiverse tropical leaf beetles (Coleoptera: Chrysomelidae). We compared patterns of elevational diversity and species composition of beetles in two types of forests (on mountain ridges and in valleys) and analyzed whether differences in the rate of species turnover among forest habitats lead to shifts in patterns of elevational diversity when scaling up from the local study site to the elevational belt level...
March 9, 2018: Oecologia
Teresita M Porter, Mehrdad Hajibabaei
We introduce a method for assigning names to CO1 metabarcode sequences with confidence scores in a rapid, high-throughput manner. We compiled nearly 1 million CO1 barcode sequences appropriate for classifying arthropods and chordates. Compared to our previous Insecta classifier, the current classifier has more than three times the taxonomic coverage, including outgroups, and is based on almost five times as many reference sequences. Unlike other popular rDNA metabarcoding markers, we show that classification performance is similar across the length of the CO1 barcoding region...
March 9, 2018: Scientific Reports
Albertas Dvirnas, Christoffer Pichler, Callum L Stewart, Saair Quaderi, Lena K Nyberg, Vilhelm Müller, Santosh Kumar Bikkarolla, Erik Kristiansson, Linus Sandegren, Fredrik Westerlund, Tobias Ambjörnsson
The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds...
2018: PloS One
Samuel T Coradetti, Dominic Pinel, Gina Geiselman, Masakazu Ito, Stephen Mondo, Morgann C Reilly, Ya-Fang Cheng, Stefan Bauer, Igor Grigoriev, John M Gladden, Blake A Simmons, Rachel Brem, Adam P Arkin, Jeffrey M Skerker
The basidiomycete yeast Rhodosporidium toruloides (a.k.a. Rhodotorula toruloides ) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains...
March 9, 2018: ELife
Rolando Robert, Kenneth Francis Rodrigues, Zarinah Waheed, Subbiah Vijay Kumar
This study is aimed at establishing a baseline on the genetic diversity of the Acropora corals of Sabah, North Borneo based on variations in the partial COI and CYB nucleotide sequences. Comparison across 50 shallow-water Acropora morphospecies indicated that the low substitution rates in the two genes were due to negative selection and that rate heterogeneity between them was asymmetric. CYB appeared to have evolved faster than COI in the Acropora as indicated by differences in the rate of pairwise genetic distance, degrees of transition bias (Ts /Tv ), synonymous-to-nonsynonymous rate ratio (dN /dS ), and substitution patterns at the three codon positions...
March 9, 2018: Mitochondrial DNA. Part A. DNA Mapping, Sequencing, and Analysis
Rômulo V Paixão, Josiane Ribolli, Evoy Zaniboni-Filho
Steindachneridion scriptum is an important species as a resource for fisheries and aquaculture; it is currently threatened and has a reduced occurrence in South America. The damming of rivers, overfishing, and contamination of freshwater environments are the main impacts on the maintenance of this species. We accessed the genetic diversity and structure of S. scriptum using the DNA barcode and control region (D-loop) sequences of 43 individuals from the Upper Uruguay River Basin (UUR) and 10 sequences from the Upper Paraná River Basin (UPR), which were obtained from GenBank...
2018: Frontiers in Genetics
Zheng-Liang Wang, Tian-Zhao Wang, Hang-Feng Zhu, Zi-Ye Wang, Xiao-Ping Yu
Ladybird beetles (Coleoptera: Coccinellidae), with broad morphological diversity, wide geographic distribution and substantial agricultural significance, are a challenging group for taxonomists and phylogenetics. As a promising tool to identify and discover new species, DNA barcoding might offer significant potential for identification, taxonomy and phylogeny of ladybird beetles. In the present study, a total of 1364 COI (cytochrome C oxidase subunit I) sequences representing 128 species from 52 genera of ladybird beetles were screened for barcoding evaluation and phylogenetic analysis...
March 8, 2018: Mitochondrial DNA. Part A. DNA Mapping, Sequencing, and Analysis
Dawn S Lin, Andrey Kan, Jerry Gao, Edmund J Crampin, Philip D Hodgkin, Shalin H Naik
A thorough understanding of cellular development is incumbent on assessing the complexities of fate and kinetics of individual clones within a population. Here, we develop a system for robust periodical assessment of lineage outputs of thousands of transient clones and establishment of bona fide cellular trajectories. We appraise the development of dendritic cells (DCs) in fms-like tyrosine kinase 3 ligand culture from barcode-labeled hematopoietic stem and progenitor cells (HSPCs) by serially measuring barcode signatures and visualize these multidimensional data using developmental interpolated t-distributed stochastic neighborhood embedding (DiSNE) time-lapse movies...
March 6, 2018: Cell Reports
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