ALBERT

Description: Analysis of RNA-seq data often detects numerous ‘non-co-linear’ (NCL) transcripts, which comprised sequence segments that are topologically inconsistent with their corresponding DNA sequences in the reference genome. However, detection of NCL transcripts involves two major challenges: removal of false positives arising from alignment artifacts and discrimination between different types of NCL transcripts ( trans -spliced, circular or fusion transcripts). Here, we developed a new NCL-transcript-detecting method (‘NCLscan’), which utilized a stepwise alignment strategy to almost completely eliminate false calls (〉98% precision) without sacrificing true positives, enabling NCLscan outperform 18 other publicly-available tools (including fusion- and circular-RNA-detecting tools) in terms of sensitivity and precision, regardless of the generation strategy of simulated dataset, type of intragenic or intergenic NCL event, read depth of coverage, read length or expression level of NCL transcript. With the high accuracy, NCLscan was applied to distinguishing between trans -spliced, circular and fusion transcripts on the basis of poly(A)- and nonpoly(A)-selected RNA-seq data. We showed that circular RNAs were expressed more ubiquitously, more abundantly and less cell type-specifically than trans -spliced and fusion transcripts. Our study thus describes a robust pipeline for the discovery of NCL transcripts, and sheds light on the fundamental biology of these non-canonical RNA events in human transcriptome.

Description: Glioma is the most common and fatal primary brain tumour with poor prognosis; however, the functional roles of miRNAs in glioma malignant progression are insufficiently understood. Here, we used an integrated approach to identify miRNA functional targets during glioma malignant progression by combining the paired expression profiles of miRNAs and mRNAs across 160 Chinese glioma patients, and further constructed the functional miRNA–mRNA regulatory network. As a result, most tumour-suppressive miRNAs in glioma progression were newly discovered, whose functions were widely involved in gliomagenesis. Moreover, three miRNA signatures, with different combinations of hub miRNAs (regulations≥30) were constructed, which could independently predict the survival of patients with all gliomas, high-grade glioma and glioblastoma. Our network-based method increased the ability to identify the prognostic biomarkers, when compared with the traditional method and random conditions. Hsa-miR-524-5p and hsa-miR-628-5p, shared by these three signatures, acted as protective factors and their expression decreased gradually during glioma progression. Functional analysis of these miRNA signatures highlighted their critical roles in cell cycle and cell proliferation in glioblastoma malignant progression, especially hsa-miR-524-5p and hsa-miR-628-5p exhibited dominant regulatory activities. Therefore, network-based biomarkers are expected to be more effective and provide deep insights into the molecular mechanism of glioma malignant progression.

Description: Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff , (M1) and Hivep1, Nfil3, Prdm1 , (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

Description: We study fluctuations in the degree-2 zonal spherical harmonic coefficient of the Earth's gravity potential, C 20 , over the period 2003–2015. This coefficient is related to the Earth's oblateness and studying its temporal variations, C 20 , can be used to monitor large-scale mass movements between high and low latitude regions. We examine C 20 inferred from six different sources, including satellite laser ranging (SLR), GRACE and global geophysical fluids models. We further include estimates that we derive from measured variations in the length-of-day (LOD), from the inversion of global crustal displacements as measured by GPS, as well as from the combination of GRACE and the output of an ocean model as described by Sun et al. We apply a sequence of trend and seasonal moving average filters to the different time-series in order to decompose them into an interannual, a seasonal and an intraseasonal component. We then perform a comparison analysis for each component, and we further estimate the noise level contained in the different series using an extended version of the three-cornered-hat method. For the seasonal component, we generally obtain a very good agreement between the different sources, and except for the LOD-derived series, we find that over 90 per cent of the variance in the seasonal components can be explained by the sum of an annual and semiannual oscillation of constant amplitudes and phases, indicating that the seasonal pattern is stable over the considered time period. High consistency between the different estimates is also observed for the intraseasonal component, except for the solution from GRACE, which is known to be affected by a strong tide-like alias with a period of about 161 d. Estimated interannual components from the different sources are generally in agreement with each other, although estimates from GRACE and LOD present some discrepancies. Slight deviations are further observed for the estimate from the geophysical models, likely to be related to the omission of polar ice and groundwater changes in the model combination we use. On the other hand, these processes do not seem to play an important role at seasonal and shorter timescales, as the sum of modelled atmospheric, oceanic and hydrological effects effectively explains the observed C 20 variations at those scales. We generally obtain very good results for the solution from SLR, and we confirm that this well-established technique accurately tracks changes in C 20 . Good agreement is further observed for the estimate from the GPS inversion, showing that this indirect method is successful in capturing fluctuations in C 20 on scales ranging from intra- to interannual. Obtaining accurate estimates from LOD, however, remains a challenging task and more reliable models of atmospheric wind fields are needed in order to obtain high-quality C 20 , in particular at the seasonal scale. The combination of GRACE data and the output of an ocean model appears to be a promising approach, particularly since corresponding C 20 is not affected by tide-like aliases, and generally gives better results than the solution from GRACE, which still seems to be of rather poor quality.

Description: Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information. Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

Description: A major challenge in the field of shotgun metagenomics is the accurate identification of organisms present within a microbial community, based on classification of short sequence reads. Though existing microbial community profiling methods have attempted to rapidly classify the millions of reads output from modern sequencers, the combination of incomplete databases, similarity among otherwise divergent genomes, errors and biases in sequencing technologies, and the large volumes of sequencing data required for metagenome sequencing has led to unacceptably high false discovery rates (FDR). Here, we present the application of a novel, gene-independent and signature-based metagenomic taxonomic profiling method with significantly and consistently smaller FDR than any other available method. Our algorithm circumvents false positives using a series of non-redundant signature databases and examines G enomic O rigins T hrough T axonomic CHA llenge (GOTTCHA). GOTTCHA was tested and validated on 20 synthetic and mock datasets ranging in community composition and complexity, was applied successfully to data generated from spiked environmental and clinical samples, and robustly demonstrates superior performance compared with other available tools.

Description: Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these ‘repressed transcripts’ have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.

Description: With read lengths of currently up to 2 x 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%.

Description: The relative gravimeter is the primary terrestrial instrument for measuring spatially and temporally varying gravitational fields. The background noise of the instrument—that is, non-linear drift and random tares—typically requires some form of least-squares network adjustment to integrate data collected during a campaign that may take several days to weeks. Here, we present an approach to remove the change in the observed relative-gravity differences caused by hydrologic or other transient processes during a single campaign, so that the adjusted gravity values can be referenced to a single epoch. The conceptual approach is an example of coupled hydrogeophysical inversion, by which a hydrologic model is used to inform and constrain the geophysical forward model. The hydrologic model simulates the spatial variation of the rate of change of gravity as either a linear function of distance from an infiltration source, or using a 3-D numerical groundwater model. The linear function can be included in and solved for as part of the network adjustment. Alternatively, the groundwater model is used to predict the change of gravity at each station through time, from which the accumulated gravity change is calculated and removed from the data prior to the network adjustment. Data from a field experiment conducted at an artificial-recharge facility are used to verify our approach. Maximum gravity change due to hydrology (observed using a superconducting gravimeter) during the relative-gravity field campaigns was up to 2.6 μGal d –1 , each campaign was between 4 and 6 d and one month elapsed between campaigns. The maximum absolute difference in the estimated gravity change between two campaigns, two months apart, using the standard network adjustment method and the new approach, was 5.5 μGal. The maximum gravity change between the same two campaigns was 148 μGal, and spatial variation in gravity change revealed zones of preferential infiltration and areas of relatively high groundwater storage. The accommodation for spatially varying gravity change would be most important for long-duration campaigns, campaigns with very rapid changes in gravity and (or) campaigns where especially precise observed relative-gravity differences are used in the network adjustment.

Description: A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 x 10 –4 insertions per genome per generation and the rate of IS homologous recombination is 4.5 x 10 –5 recombinations per genome per generation. These events are mostly contributed by the IS elements IS 1 , IS 2 , IS 5 and IS 186 . Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS 186 target-sites, 5'-GGGG(N6/N7)CCCC-3'. We also detected 48 long deletions not involving IS elements.