Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling

BACKGROUND:Massively parallel sequencing offers an enormous potential for expression profiling, in particular for interspecific comparisons. Currently, different platforms for massively parallel sequencing are available, which differ in read length and sequencing costs. The 454-technology offers the highest read length. The other sequencing technologies are more cost effective, on the expense of shorter reads. Reliable expression profiling by massively parallel sequencing depends crucially on the accuracy to which the reads could be mapped to the corresponding genes. METHODOLOGY/PRINCIPAL FINDINGS:We performed an in silico analysis to evaluate whether incorrect mapping of the sequence reads results in a biased expression pattern. A comparison of six available mapping software tools indicated a considerable heterogeneity in mapping speed and accuracy. Independently of the software used to map the reads, we found that for compact genomes both short (35 bp, 50 bp) and long sequence reads (100 bp) result in an almost unbiased expression pattern. In contrast, for species with a larger genome containing more gene families and repetitive DNA, shorter reads (35-50 bp) produced a considerable bias in gene expression. In humans, about 10% of the genes had fewer than 50% of the sequence reads correctly mapped. Sequence polymorphism up to 9% had almost no effect on the mapping accuracy of 100 bp reads. For 35 bp reads up to 3% sequence divergence did not affect the mapping accuracy strongly. The effect of indels on the mapping efficiency strongly depends on the mapping software. CONCLUSIONS/SIGNIFICANCE:In complex genomes, expression profiling by massively parallel sequencing could introduce a considerable bias due to incorrectly mapped sequence reads if the read length is short. Nevertheless, this bias could be accounted for if the genomic sequence is known. Furthermore, sequence polymorphisms and indels also affect the mapping accuracy and may cause a biased gene expression measurement. The choice of the mapping software is highly critical and the reliability depends on the presence/absence of indels and the divergence between reads and the reference genome. Overall, we found SSAHA2 and CLC to produce the most reliable mapping results

Translate to divide: сontrol of the cell cycle by protein synthesis

Protein synthesis underpins much of cell growth and, consequently, cell multiplication. Understanding how proliferating cells commit and progress into the cell cycle requires knowing not only which proteins need to be synthesized, but also what determines their rate of synthesis during cell division.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

MutS and MutL Are Dispensable for Maintenance of the Genomic Mutation Rate in the Halophilic Archaeon Halobacterium salinarum NRC-1

BACKGROUND: The genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans. METHODOLOGY/PRINCIPAL FINDINGS: We calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate. CONCLUSIONS/SIGNIFICANCE: We established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs

A GATEWAY™ Destination Vector For High-Throughput Construction of Neurospora crassa histidine-3 Gene Replacement Plasmids

We report the construction of a Destination Vector, called pJHAM007, for the targeted integration of DNA sequences at the histidine-3 (his-3) locus of Neurospora crassa. pJHAM007 has all the necessary features required to perform a simple, rapid and efficient GATEWAY™ recombinational cloning with an Entry Clone to yield a his-3-gene replacement Destination Vector

A Systems Biology Approach Uncovers Cellular Strategies Used by Methylobacterium extorquens AM1 During the Switch from Multi- to Single-Carbon Growth

When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap.This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints

Genome-Wide Maps of Mononucleosomes and Dinucleosomes Containing Hyperacetylated Histones of Aspergillus fumigatus

It is suggested that histone modifications and/or histone variants influence the nucleosomal DNA length. We sequenced both ends of mononucleosomal and dinucleosomal DNA fragments of the filamentous fungus Aspergillus fumigatus, after treatment with the histone deacetylase inhibitor trichostatin A (TSA). After mapping the DNA fragments to the genome, we identified >7 million mononucleosome positions and >7 million dinucleosome positions. We showed that the distributions of the lengths of the mononucleosomal DNA fragments after 15-min and 30-min treatments with micrococcal nuclease (MNase) showed a single peak at 168 nt and 160 nt, respectively. The distributions of the lengths of the dinucleosomal DNA fragments after 15-min- and 30-min-treatment with MNase showed a single peak at 321 nt and 306 nt, respectively. The nucleosomal DNA fragments obtained from the TSA-treated cells were significantly longer than those obtained from the untreated cells. On the other hand, most of the genes did not undergo any change after treatment. Between the TSA-treated and untreated cells, only 77 genes had ≥2-fold change in expression levels. In addition, our results showed that the locations where mononucleosomes were frequently detected were conserved between the TSA-treated cells and untreated cells in the gene promoters (lower density of the nucleosomes). However, these locations were less conserved in the bodies (higher density of the nucleosomes) of genes with ≥2-fold changes. Our findings indicate that TSA influences the nucleosome positions, especially of the regions with high density of the nucleosomes by elongation of the nucleosomal DNA. However, most of the nucleosome positions are conserved in the gene promoters, even after treatment with TSA, because of the low density of nucleosomes in the gene promoters

Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

BACKGROUND:Metazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3' end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3' end processing. The 3' end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS:In Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3' end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells. CONCLUSIONS/SIGNIFICANCE:These data are consistent with a model in which changing the 3' end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development

rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events

Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen