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Single-cell genomics

Charles Cole, Ashley Byrne, Anna E Beaudin, E Camilla Forsberg, Christopher Vollmers
RNA-sequencing (RNA-seq) is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase-based Smart-seq2 protocol to create RNA-seq libraries that capture the 5' end of transcripts. The Tn5Prime method dramatically streamlines the 5' capture process and is both cost effective and reliable...
March 14, 2018: Nucleic Acids Research
Peter Vegh, Muzlifah Haniffa
Application of single-cell genomics technologies has revolutionized our approach to study the immune system. Unravelling the functional diversity of immune cells and their coordinated response is key to understanding immunity. Single-cell transcriptomics technologies provide high-dimensional assessment of the transcriptional states of immune cells and have been successfully applied to discover new immune cell types, reveal haematopoietic lineages, identify gene modules dictating immune responses and investigate lymphocyte antigen receptor diversity...
March 14, 2018: Briefings in Functional Genomics
Radoslav Aleksandrov, Anton Dotchev, Ina Poser, Dragomir Krastev, Georgi Georgiev, Greta Panova, Yordan Babukov, Georgi Danovski, Teodora Dyankova, Lars Hubatsch, Aneliya Ivanova, Aleksandar Atemin, Marina N Nedelcheva-Veleva, Susanne Hasse, Mihail Sarov, Frank Buchholz, Anthony A Hyman, Stephan W Grill, Stoyno S Stoynov
A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites...
March 15, 2018: Molecular Cell
Jeffrey Zielich, Elena Tzima, Eva Ayla Schröder, Faten Jemel, Barbara Conradt, Eric J Lambie
P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson's disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity; however, the identity of the transport substrate(s) remains unknown...
2018: PloS One
Amporn Suphatrakul, Thaneeya Duangchinda, Natapong Jupatanakul, Kanjanawadee Prasittisa, Suppachoke Onnome, Jutharat Pengon, Bunpote Siridechadilok
Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses...
2018: PloS One
Te-Sha Tsai, Sonika Tyagi, Justin C St John
STUDY QUESTION: What are the molecular differences between mitochondrial DNA (mtDNA)-deficient and mtDNA-normal oocytes and how does mitochondrial supplementation alter these? SUMMARY ANSWER: Changes to DNA methylation in a 5' cytosine-phosphate-guanine 3' (CpG) island in the mtDNA-specific replication factor (DNA polymerase gamma (POLG)) of mtDNA-deficient oocytes mediates an increase in mtDNA copy number by the 2-cell stage that positively modulates the expression of nuclear genes, which affect cellular and metabolic processes, following autologous mitochondrial supplementation...
March 13, 2018: Human Reproduction
Qingqing Wei, Ruiqi Li, Liang Zhong, Haiyuan Mu, Shaopeng Zhang, Liang Yue, Jinzhu Xiang, Enhong Li, Minglei Zhi, Suying Cao, Jianyong Han
After zygotic genome activation and lineage specification, zygotes develop into late blastocysts comprising three distinct cell types. The molecular mechanisms underlying this progress are largely unknown in pigs. Here, we intended to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the porcine blastocysts. Using a quantitative microfluidics approach in single cells, we detected mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency simultaneously in 480 individual cells derived from porcine preimplantation embryos...
March 13, 2018: Biology of Reproduction
Qing Mao, Robert Chin, Weiwei Xie, Yuqing Deng, Wenwei Zhang, Huixin Xu, Rebecca Yu Zhang, Quan Shi, Erin E Peters, Natali Gulbahce, Zhenyu Li, Fang Chen, Radoje Drmanac, Brock A Peters
BACKGROUND: Amniocentesis is a common procedure, the primary purpose of which is to collect cells from the fetus to allow testing for abnormal chromosomes, altered chromosomal copy number, or a small number of genes that have small single- to multibase defects. Here we demonstrate the feasibility of generating an accurate whole-genome sequence of a fetus from either the cellular or cell-free DNA (cfDNA) of an amniotic sample. METHODS: cfDNA and DNA isolated from the cell pellet of 31 amniocenteses were sequenced to approximately 50× genome coverage by use of the Complete Genomics nanoarray platform...
March 15, 2018: Clinical Chemistry
Suhyung Cho, Donghui Choe, Eunju Lee, Sun Chang Kim, Bernhard Ø Palsson, Byung-Kwan Cho
Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq)...
March 15, 2018: ACS Synthetic Biology
Cosimo Cumbo, Luciana Impera, Crescenzio Francesco Minervini, Paola Orsini, Luisa Anelli, Antonella Zagaria, Nicoletta Coccaro, Giuseppina Tota, Angela Minervini, Paola Casieri, Claudia Brunetti, Antonella Russo Rossi, Elisa Parciante, Giorgina Specchia, Francesco Albano
For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology...
February 16, 2018: Oncotarget
Yan Hao, Dawei Chen, Zhiguo Zhang, Ping Zhou, Yunxia Cao, Zhaolian Wei, Xiaofeng Xu, Beili Chen, Weiwei Zou, Mingrong Lv, Dongmei Ji, Xiaojin He
Hearing loss may place a heavy burden on the patient and patient's family. Given the high incidence of hearing loss among newborns and the huge cost of treatment and care (including cochlear implantation), prenatal diagnosis is strongly recommended. Termination of the fetus may be considered as an extreme outcome to the discovery of a potential deaf fetus, and therefore preimplantation genetic diagnosis has become an important option for avoiding the birth of affected children without facing the risk of abortion following prenatal diagnosis...
April 2018: Oncology Letters
Warren Winick-Ng, R Jane Rylett
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by synapse dysfunction and cognitive impairment. Understanding the development and progression of AD is challenging, as the disease is highly complex and multifactorial. Both environmental and genetic factors play a role in AD pathogenesis, highlighted by observations of complex DNA modifications at the single gene level, and by new evidence that also implicates changes in genome architecture in AD patients. The four-dimensional structure of chromatin in space and time is essential for context-dependent regulation of gene expression in post-mitotic neurons...
2018: Frontiers in Molecular Neuroscience
María Cecilia Gimenez, Flavia Adriana Zanetti, Mauricio R Terebiznik, María Isabel Colombo, Laura Ruth Delgui
Birnaviruses are unconventional members of the double-stranded RNA (dsRNA) viruses group that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the viral encoded RNA-dependent RNA-polymerase (RdRp). This and other structural features suggests that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses...
March 14, 2018: Journal of Virology
Mathew A Cherian, Sydney Olson, Hemalatha Sundaramoorthi, Kitra Cates, Xiaogang Cheng, John Harding, Andrew Martens, Grant A Challen, Manoj Tyagi, Lee Ratner, Daniel Rauch
The human T cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, likely as a result of specific immuno-editing, Tax expression is downregulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL, and the K59R mutation is the most common single-nucleotide variation in IRF4 and is found exclusively in ATL...
March 14, 2018: Journal of Biological Chemistry
Darren A Cusanovich, James P Reddington, David A Garfield, Riza M Daza, Delasa Aghamirzaie, Raquel Marco-Ferreres, Hannah A Pliner, Lena Christiansen, Xiaojie Qiu, Frank J Steemers, Cole Trapnell, Jay Shendure, Eileen E M Furlong
Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila melanogaster embryos spanning three landmark embryonic stages: 2-4 h after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises around 6,000 multipotent cells; 6-8 h after egg laying (predominantly stage 10-11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying (predominantly stage 13), when each of the embryo's more than 20,000 cells are undergoing terminal differentiation...
March 14, 2018: Nature
Gregory A Babbitt, Jamie S Mortensen, Erin E Coppola, Lily E Adams, Justin K Liao
Traditional informatics in comparative genomics work only with static representations of biomolecules (i.e., sequence and structure), thereby ignoring the molecular dynamics (MD) of proteins that define function in the cell. A comparative approach applied to MD would connect this very short timescale process, defined in femtoseconds, to one of the longest in the universe: molecular evolution measured in millions of years. Here, we leverage advances in graphics-processing-unit-accelerated MD simulation software to develop a comparative method of MD analysis and visualization that can be applied to any two homologous Protein Data Bank structures...
March 13, 2018: Biophysical Journal
Sellappan Selvaraju, Sivashanmugam Parthipan, Lakshminarayana Somashekar, B Krishnan Binsila, Atul P Kolte, Arunachalam Arangasamy, Janivara Parameshwaraiah Ravindra, Stephen A Krawetz
With artificial insemination (AI) and other precision dependent assisted reproductive technologies (ART) being followed in large scale in human and animal reproduction, assessing semen quality and fertilizability is under continuous scrutiny. Various tests have been developed to predict semen quality, but so far no single, highly reliable test is available. In this regard, transcriptomic profiling of spermatozoa assumes significance as it carries the information about spermatogenesis, sperm function, and paternal roles in post-fertilization events...
March 14, 2018: Systems Biology in Reproductive Medicine
Peter Sykora, Ylenia Chiari, Andrew Heaton, Nickolas Moreno, Scott Glaberman, Robert W Sobol
DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage...
March 14, 2018: Environmental and Molecular Mutagenesis
Yungang Xu, Xiaobo Zhou
Single-cell sequencing interrogates the sequence or chromatin information from individual cells with advanced next-generation sequencing technologies. It provides a higher resolution of cellular differences and a better understanding of the underlying genetic and epigenetic mechanisms of an individual cell in the context of its survival and adaptation to microenvironment. However, it is more challenging to perform single-cell sequencing and downstream data analysis, owing to the minimal amount of starting materials, sample loss, and contamination...
2018: Methods in Molecular Biology
Reinier Snetselaar, Matthijs F M van Oosterhout, Jan C Grutters, Coline H M van Moorsel
The enzyme telomerase reverse transcriptase (TERT) is essential for telomere maintenance. In replicating cells, maintenance of telomere length is important for the preservation of vital genetic information and prevention of genomic instability. A common genetic variant in TERT , rs2736100 C/A, is associated with both telomere length and multiple diseases. Carriage of the C allele is associated with longer telomere length, while carriage of the A allele is associated with shorter telomere length. Furthermore, some diseases have a positive association with the C and some with the A allele...
2018: Frontiers in Medicine
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