BackgroundRNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally, causing some to be overrepresented. Unique molecular identifiers (UMIs) can be used to distinguish undesirable PCR duplicates derived from a single molecule and identical but biologically meaningful reads from different molecules.ResultsWe have incorporated UMIs into RNA-seq and small RNA-seq protocols and developed tools to analyze the resulting data. Our UMIs contain stretches of random nucleotides whose lengths sufficiently capture diverse molecule species in both RNA-seq and small RNA-seq libraries generated from mouse testis. Our approach yields high-quality data while allowing unique tagging of all molecules in high-depth libraries.ConclusionsUsing simulated and real datasets, we demonstrate that our methods increase the reproducibility of RNA-seq and small RNA-seq data. Notably, we find that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine PCR duplicate frequency. Finally, we show that computational removal of PCR duplicates based only on their mapping coordinates introduces substantial bias into data analysis.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4933-1) contains supplementary material, which is available to authorized users.

Pachytene piRNAs, which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like miRNAs, cleave targets like siRNAs, or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 ( pi6 ) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6 −/− fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving mRNAs encoding proteins required for sperm function, pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10 as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.

Transposable elements (TEs) have a major impact on genome evolution, but they are potentially deleterious, and most of them are silenced by epigenetic mechanisms, such as DNA methylation. Here, we report the characterization of a TE encoding an activity to counteract epigenetic silencing by the host. In Arabidopsis thaliana, we identified a mobile copy of the Mutator‐like element (MULE) with degenerated terminal inverted repeats (TIRs). This TE, named Hiun (Hi), is silent in wild‐type plants, but it transposes when DNA methylation is abolished. When a Hi transgene was introduced into the wild‐type background, it induced excision of the endogenous Hi copy, suggesting that Hi is the autonomously mobile copy. In addition, the transgene induced loss of DNA methylation and transcriptional activation of the endogenous Hi. Most importantly, the trans‐activation of Hi depends on a Hi‐encoded protein different from the conserved transposase. Proteins related to this anti‐silencing factor, which we named VANC, are widespread in the non‐TIR MULEs and may have contributed to the recent success of these TEs in natural Arabidopsis populations.

Duck astroviruses (DAstVs) are known to cause duck viral hepatitis; however, little is known regarding their molecular biology. Here, we report the complete sequence of a DAstV associated with a recent outbreak of fatal hepatitis in ducklings in China. Sequence analyses indicated that the genome of DAstV possessed a typical astrovirus organization and also exhibited two unique features. The polyadenylated genome comprised 7722 nt, which is the largest among astroviruses sequenced to date. The ORF2 of DAstV was not in the same reading frame as either ORF1a or ORF1b, which was distinct from all other astroviruses. Sequence comparisons and phylogenetic analyses revealed that DAstV was more closely related to turkey astrovirus (TAstV) type 2, TAstV-3 and TAstV/MN/01 (a possible new TAstV serotype) than to TAstV-1 or other astroviruses. These findings suggest that astroviruses may transmit across ducks and turkeys.Astroviruses are non-enveloped, single-stranded, positivesense RNA viruses, which have been demonstrated to infect both mammalian and avian hosts (Monroe et al., 2005). The genomes of these viruses range in size from 6.1 to 7.3 kb, consisting of three ORFs (1a, 1b, and 2) (Finkbeiner et al., 2008). All astroviruses share a rather conserved frameshift slippery sequence in the overlap region between ORF1a and ORF1b, which directs the synthesis of an ORFla/lb fusion polyprotein (Jiang et al., 1993;Lewis et al., 1994;Lewis & Matsui, 1996;Marczinke et al., 1994). ORF1a and ORF1b encode the non-structural proteins, which include several transmembrane helical motifs, a serine protease, a nuclear localization signal (NLS), and an RNA-dependent RNA polymerase (RdRp) motif (Jiang et al., 1993;Lewis et al., 1994;Willcocks et al., 1994). ORF2 encodes the capsid protein that is required for virion formation.Astroviruses in ducks have been associated with a fatal hepatitis which occurred in the UK, historically known as duck hepatitis virus type 2 (DHV-2) (Asplin, 1965; Gough et al., 1984Gough et al., , 1985. Originally thought to be a picornavirus, DHV-2 was later characterized as an astrovirus by morphology and renamed duck astrovirus 1 (DAstV-1) (Gough et al., 1984(Gough et al., , 1985Monroe et al., 2005). DHV-1 and a later described serotype 3 (DHV-3), isolated in the USA (Haider & Calnek, 1979), are still classified as picornaviruses (Stanway et al., 2005). Recently, DHV-1 was confirmed as a picornavirus belonging to a novel genus (Ding & Zhang, 2007;Kim et al., 2006;. It has also been proposed that DHV-1 be renamed duck hepatitis A virus (DHAV) genotype A (DHAV-A) and that two other newly discovered DHV serotypes (Kim et al., 2007; be designated DHAV-B and DHAV-C (Fu et al., 2008;Wang et al., 2008). More recently, a 391 nt RdRp sequence has been determined for DAstV-1 and DHV-3 and comparisons indicate that they are both astroviruses (Todd et al., 2009). In this study, we report the complete genomic sequence of a DAstV strain associated with a fatal hepatitis which occurred in China in 2008. The genomic features ...

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest, Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families reveal T. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, and T. ni siRNAs are not 2´-O-methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. The T. ni genome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.

ENCODE 3 (2012-2017) expanded production and added new types of assays 8 (Fig. 1, Extended Data Fig. 1), which revealed landscapes of RNA binding and the 3D organization of chromatin via methods such as chromatin interaction analysis by paired-end tagging (ChIA-PET) and Hi-C chromosome conformation capture. Phases 2 and 3 delivered 9,239 experiments (7,495 in human and 1,744 in mouse) in more than 500 cell types and tissues, including mapping of transcribed regions and transcript isoforms, regions of transcripts recognized by RNA-binding proteins, transcription factor binding regions, and regions that harbour specific histone modifications, open chromatin, and 3D chromatin interactions. The results of all of these experiments are available at the ENCODE portal (http://www.encodeproject.org). These efforts, combined with those of related projects and many other laboratories, have produced a greatly enhanced view of the human genome (Fig. 2), identifying 20,225 protein-coding and 37,595 noncoding genes

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A simple and rapid method for detecting chemical components of individual aerosol particles on Klarite substrate with surface-enhanced Raman spectroscopy (SERS) is described. For both single simulated aerosol particles and ambient atmospheric particles, this new analytical method promotes the enhancement factor of the Raman signal. The spectra of ammonium sulfate and naphthalene particles at the microscopic level are enhanced by a factor of 6 and therefore greatly improve the detection of the chemical composition of an individual aerosol particle. When aerosol particles are found over a microscopic domain, a set of Raman spectra with chemical information can be obtained via SERS mapping. The maps illustrate the distribution of organic or inorganic species on the SERS substrate. This constitutes a facile and rapid method to study aerosol particles. This new method allows the analysis of chemical composition in single aerosol particles, demonstrating the power of SERS to probe the ambient atmospheric particles and to study the formation of aerosol particles.