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Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state-of-the-art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria, and chloroplasts or other plastids. By examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, that is, the one following the initial methionine, has a strong influence on the classification. We observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position 2, compared with 20% in other plant proteins. We also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide. The importance of this feature for predictions has not been highlighted before., QC 20191126

Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister Glade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this subclade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses, QC 20181126

Eukaryotic cellular programs are context dependent. Furthermore, gene regulation exerts this control through layers of interacting molecules. Therefore, genetic variation could interfere in the way these molecules behave by introducing mutations in their underlying DNA sequence. This phenomenon may also selectively affect some gene copies, hence producing an Allele Specific Expression (ASE). This work investigates the effect of genetic variation on genes exhibiting ASE to identify variants with potential regulatory role. Consequently, it shortlists and characterizes variants near protein coding regions as well as introns. Moreover, it relies on analyzing RNA-sequencing and SNP-array data of primary white blood cells of eight European healthy individuals. The study also evaluates two sets of samples: the cells in their basal state and after treatment with lipopolysaccharide (LPS). The proposed model shortlists exon variants representative of ASE, then pairs them against intron/upstream mutations to process linkage disequilibrium analysis. Finally, those which co-segregate with a correlation greater than 0.80 are selected as potential intron/upstream regulatory variants. This process yielded 546 intronic and 80 upstream variants of which 28.20% and 31.25% corresponded to known regulatory elements, according to the Variant Effect Predictor from ENSEMBL. Furthermore, the selected variants are enriched for terms describing an immune response. This trait is especially true for the LPS samples that indeed reacted as if under a bacterial infection. Finally, the selected upstream variants occur more often proximal to the core promoter than to the upper limit of 10kb.

Allele-specific expression (ASE) is the imbalance in transcription between maternal and paternal alleles at a locus and can be probed in single individuals using massively parallel DNA sequencing technology. Assessing ASE within a single sample provides a static picture of the ASE, but the magnitude of ASE for a given transcript may vary between different biological conditions in an individual. Such condition-dependent ASE could indicate a genetic variation with a functional role in the phenotypic difference. We investigated ASE through RNA-sequencing of primary white blood cells from eight human individuals before and after the controlled induction of an inflammatory response, and detected condition-dependent and static ASE at 211 and 13021 variants, respectively. We developed a method, GeneiASE, to detect genes exhibiting static or condition-dependent ASE in single individuals. GeneiASE performed consistently over a range of read depths and ASE effect sizes, and did not require phasing of variants to estimate haplotypes. We observed condition-dependent ASE related to the inflammatory response in 19 genes, and static ASE in 1389 genes. Allele-specific expression was confirmed by validation of variants through real-time quantitative RT-PCR, with RNA-seq and RT-PCR ASE effect-size correlations r = 0.67 and r = 0.94 for static and condition-dependent ASE, respectively., QC 20160309

RNA sequencing has become widely used in gene expression profiling experiments. Prior to any RNA sequencing experiment the quality of the RNA must be measured to assess whether or not it can be used for further downstream analysis. The RNA integrity number (RIN) is a scale used to measure the quality of RNA that runs from 1 (completely degraded) to 10 (intact). Ideally, samples with high RIN (>8) are used in RNA sequencing experiments. RNA, however, is a fragile molecule which is susceptible to degradation and obtaining high quality RNA is often hard, or even impossible when extracting RNA from certain clinical tissues. Thus, occasionally, working with low quality RNA is the only option the researcher has. Here we investigate the effects of RIN on RNA sequencing and suggest a computational method to handle data from samples with low quality RNA which also enables reanalysis of published datasets. Using RNA from a human cell line we generated and sequenced samples with varying RINs and illustrate what effect the RIN has on the basic procedure of RNA sequencing; both quality aspects and differential expression. We show that the RIN has systematic effects on gene coverage, false positives in differential expression and the quantification of duplicate reads. We introduce 3' tag counting (3TC) as a computational approach to reliably estimate differential expression for samples with low RIN. We show that using the 3TC method in differential expression analysis significantly reduces false positives when comparing samples with different RIN, while retaining reasonable sensitivity., QC 20140423

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C(0)t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C(0)t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found., Updated from manuscript to article in journal.QC 20140912

Background: Strand specific RNA sequencing is rapidly replacing conventional cDNA sequencing as an approach for assessing information about the transcriptome. Alongside improved laboratory protocols the development of bioinformatical tools is steadily progressing. In the current procedure the Illumina TruSeq library preparation kit is used, along with additional reagents, to make stranded libraries in an automated fashion which are then sequenced on Illumina HiSeq 2000. By the use of freely available bioinformatical tools we show, through quality metrics, that the protocol is robust and reproducible. We further highlight the practicality of strand specific libraries by comparing expression of strand specific libraries to non-stranded libraries, by looking at known antisense transcription of pseudogenes and by identifying novel transcription. Furthermore, two ribosomal depletion kits, RiboMinus and RiboZero, are compared and two sequence aligners, Tophat2 and STAR, are also compared. Results: The, non-stranded, Illumina TruSeq kit can be adapted to generate strand specific libraries and can be used to access detailed information on the transcriptome. The RiboZero kit is very effective in removing ribosomal RNA from total RNA and the STAR aligner produces high mapping yield in a short time. Strand specific data gives more detailed and correct results than does non-stranded data as we show when estimating expression values and in assembling transcripts. Even well annotated genomes need improvements and corrections which can be achieved using strand specific data. Conclusions: Researchers in the field should strive to use strand specific data; it allows for more confidence in the data analysis and is less likely to lead to false conclusions. If faced with analysing non-stranded data, researchers should be well aware of the caveats of that approach., QC 20150317

Melanoma of the eye is a rare and distinct subtype of melanoma, which only rarely are familial. However, cases of uveal melanoma (UM) have been found in families with mixed cancer syndromes. Here, we describe a comprehensive search for inherited genetic variation in a family with multiple cases of UM but no aggregation of other cancer diagnoses. The proband is a woman diagnosed with UM at 16 years who within 6 months developed liver metastases. We also identified two older paternal relatives of the proband who had died from UM. We performed exome sequencing of germline DNA from members of the affected family. Exome-wide analysis identified a novel loss-of-function mutation in the BAP1 gene, previously suggested as a tumor suppressor. The mutation segregated with the UM phenotype in this family, and we detected a loss of the wild-type allele in the UM tumor of the proband, strongly supporting a causative association with UM. Screening of BAP1 germline mutations in families predisposed for UM may be used to identify individuals at increased risk of disease. Such individuals may then be enrolled in preventive programs and regular screenings to facilitate early detection and thereby improve prognosis., QC 20130325

Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A(+)) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce., QC 20130305

Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS). To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches - gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of upregulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/-LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin, QC 20120515

Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein-coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq data is necessary to minimize length bias., QC 20110119

Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for sugge, QC 20111206