Pathway-based genetic analysis of preterm birth

Preterm birth in the United States is now 12%. Multiple genes, gene networks, and variants have been associated with this disease. Using a custom database for preterm birth (dbPTB) with a refined set of genes extensively curated from literature and biological databases, we analyzed GWAS of preterm birth for complete genotype data on nearly 2000 preterm and term mothers. We used both the curated genes and a genome-wide approach to carry out a pathway-based analysis. There were 19 significant pathways, which withstood FDR correction for multiple testing that were identified using both the curated genes and the genome-wide approach. The analysis based on the curated genes was more significant than genome-wide in 15 out of 19 pathways. This approach demonstrates the use of a validated set of genes, in the analysis of otherwise unsuccessful GWAS data, to identify gene–gene interactions in a way that enhances statistical power and discovery.     

OpenDMAP: An open source, ontology-driven concept analysis engine, with applications to capturing knowledge regarding protein transport, protein interactions and cell-type-specific gene expression

Background

Microarray technology provides an efficient means for globally exploring physiological processes governed by the coordinated expression of multiple genes. However, identification of genes differentially expressed in microarray experiments is challenging because of their potentially high type I error rate. Methods for large-scale statistical analyses have been developed but most of them are applicable to two-sample or two-condition data.

Results

We developed a large-scale multiple-group F-test based method, named ranking analysis of F-statistics (RAF), which is an extension of ranking analysis of microarray data (RAM) for two-sample t-test. In this method, we proposed a novel random splitting approach to generate the null distribution instead of using permutation, which may not be appropriate for microarray data. We also implemented a two-simulation strategy to estimate the false discovery rate. Simulation results suggested that it has higher efficiency in finding differentially expressed genes among multiple classes at a lower false discovery rate than some commonly used methods. By applying our method to the experimental data, we found 107 genes having significantly differential expressions among 4 treatments at <0.7% FDR, of which 31 belong to the expressed sequence tags (ESTs), 76 are unique genes who have known functions in the brain or central nervous system and belong to six major functional groups.

Conclusion

Our method is suitable to identify differentially expressed genes among multiple groups, in particular, when sample size is small.     

Evaluation and optimisation of indel detection workflows for ion torrent sequencing of the BRCA1 and BRCA2 genes

Background

The Ion Torrent PGM is a popular benchtop sequencer that shows promise in replacing conventional Sanger sequencing as the gold standard for mutation detection. Despite the PGM’s reported high accuracy in calling single nucleotide variations, it tends to generate many false positive calls in detecting insertions and deletions (indels), which may hinder its utility for clinical genetic testing.

Results

Recently, the proprietary analytical workflow for the Ion Torrent sequencer, Torrent Suite (TS), underwent a series of upgrades. We evaluated three major upgrades of TS by calling indels in the BRCA1 and BRCA2 genes. Our analysis revealed that false negative indels could be generated by TS under both default calling parameters and parameters adjusted for maximum sensitivity. However, indel calling with the same data using the open source variant callers, GATK and SAMtools showed that false negatives could be minimised with the use of appropriate bioinformatics analysis. Furthermore, we identified two variant calling measures, Quality-by-Depth (QD) and VARiation of the Width of gaps and inserts (VARW), which substantially reduced false positive indels, including non-homopolymer associated errors without compromising sensitivity. In our best case scenario that involved the TMAP aligner and SAMtools, we achieved 100% sensitivity, 99.99% specificity and 29% False Discovery Rate (FDR) in indel calling from all 23 samples, which is a good performance for mutation screening using PGM.

Conclusions

New versions of TS, BWA and GATK have shown improvements in indel calling sensitivity and specificity over their older counterpart. However, the variant caller of TS exhibits a lower sensitivity than GATK and SAMtools. Our findings demonstrate that although indel calling from PGM sequences may appear to be noisy at first glance, proper computational indel calling analysis is able to maximize both the sensitivity and specificity at the single base level, paving the way for the usage of this technology for future clinical genetic testing.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-516) contains supplementary material, which is available to authorized users.     

Convergent origins and rapid evolution of spliced leader trans-splicing in Metazoa: Insights from the Ctenophora and Hydrozoa

Replacement of mRNA 5′ UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3′ of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Detrimental effects of an autosomal selfish genetic element on sperm competitiveness in house mice

Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males. We used a vertebrate model species to test whether females can avoid an autosomal SGE, the t haplotype, through polyandry. The t haplotype in house mice exhibits strong drive in t heterozygous males by affecting spermatogenesis and is associated with homozygous in utero lethality. We used controlled matings to test the effect of the t haplotype on sperm competitiveness. Regardless of mating order, t heterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of the t haplotype on sperm quality. We provide, to our knowledge, the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry.     

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii,Ae. triuncialis and Ae. cylindrica,and their molecular organization

Background and Aims Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U^tU^tC^tC^t) and Ae. cylindrica (D^cD^cC^cC^c) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting.Methods The flow karyotypes obtained after the analysis of 4'',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA.Key Results FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C^t, T6U^tS.6U^tL-5C^tL, 1C^c and 5D^c could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2–5. This identified a partial wheat–C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C–2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected.Conclusions The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.     

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia

Background

Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.

Results

Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.

Conclusions

Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1698-7) contains supplementary material, which is available to authorized users.     

Testing the neutral theory of molecular evolution using genomic data: a comparison of the human and bovine transcriptome

Despite growing evidence of rapid evolution in protein coding genes, the contribution of positive selection to intra- and interspecific differences in protein coding regions of the genome is unclear. We attempted to see if genes coding for secreted proteins and genes with narrow expression, specifically those preferentially expressed in the mammary gland, have diverged at a faster rate between domestic cattle (Bos taurus) and humans (Homo sapiens) than other genes and whether positive selection is responsible. Using a large data set, we identified groups of genes based on secretion and expression patterns and compared them for the rate of nonsynonymous (dN) and synonymous (dS) substitutions per site and the number of radical (Dr) and conservative (Dc) amino acid substitutions. We found evidence of rapid evolution in genes with narrow expression, especially for those expressed in the liver and mammary gland and for genes coding for secreted proteins. We compared common human polymorphism data with human-cattle divergence and found that genes with high evolutionary rates in human-cattle divergence also had a large number of common human polymorphisms. This argues against positive selection causing rapid divergence in these groups of genes. In most cases dN/dS ratios were lower in human-cattle divergence than in common human polymorphism presumably due to differences in the effectiveness of purifying selection between long-term divergence and short-term polymorphism.     

Testing for differentially expressed genes with microarray data

This paper compares the type I error and power of the one- and two-sample t-tests, and the one- and two-sample permutation tests for detecting differences in gene expression between two microarray samples with replicates using Monte Carlo simulations. When data are generated from a normal distribution, type I errors and powers of the one-sample parametric t-test and one-sample permutation test are very close, as are the two-sample t-test and two-sample permutation test, provided that the number of replicates is adequate. When data are generated from a t-distribution, the permutation tests outperform the corresponding parametric tests if the number of replicates is at least five. For data from a two-color dye swap experiment, the one-sample test appears to perform better than the two-sample test since expression measurements for control and treatment samples from the same spot are correlated. For data from independent samples, such as the one-channel array or two-channel array experiment using reference design, the two-sample t-tests appear more powerful than the one-sample t-tests.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

Putative Virulence Genes and Biofilm Production Among Typical Enteroaggregative Escherichia coli Isolates from Diarrhoeic Children in Kashmir and Andhra Pradesh

Fifty-eight typical EAEC isolates from children with diarrhoea were examined for HEp-2 cell adherence assay, presence of dispersin (aap), yersiniabactin (irp2), plasmid encoded toxins (pet), Shigella enterotoxin1 (set1A) and cryptic open reading frame (shf) putative virulence genes by polymerase chain reaction as well as for biofilm production. All the isolates showed aggregative adherence pattern on HEp-2 cells. All but five isolates (91.3 %) carried aap gene. While irp2, pet, set1A and shf genes were detected in 68.9, 5.1, 39.6, and 60.3 % isolates, respectively. Thirty-three (64.7 %) isolates out of 51 tested were found to produce biofilm which was found to be significantly associated only with set1A virulence gene (P = 0.025). Highest amount of biofilm was produced by a strain that possessed all the genes studied. Out of 14 isolates in which the most frequent gene combination (aap, irp2 and shf) was observed, only six produced biofilm. It is concluded that there is significant heterogeneity in putative virulence genes of EAEC isolates from diarrhoeic children and biofilm formation is associated with multiple genes.     

Nest fidelity is driven by multi-scale information in a long-lived seabird

Although the reproductive success of most organisms depends on factors acting at several spatial scales, little is known about how organisms are able to synthesize multi-scale information to optimize reproduction. Using longitudinal data from a long-lived seabird, Monteiro''s storm-petrel, we show that average breeding success is strongly related to oceanic conditions at the population level, and we postulate that (i) individuals use proximal information (their own reproduction outcome in year t) to assess the qualities of their mate and nest and to decide to retain them or not in year t + 1; (ii) the intensity of these responses depends on the quality of the oceanic environment in year t, which affects the predictability of reproduction outcome in year t + 1. Our results confirm that mate and nest fidelities are higher following successful reproduction and that the relationship between the success of a given pair and subsequent nest fidelity is stronger in years with unfavourable oceanic conditions, suggesting that individuals rely on distant information to modulate their use of proximal information and adjust their breeding strategy.     

Phylogenetic relationships among arecoid palms (Arecaceae: Arecoideae)

Background and Aims

The Arecoideae is the largest and most diverse of the five subfamilies of palms (Arecaceae/Palmae), containing >50 % of the species in the family. Despite its importance, phylogenetic relationships among Arecoideae are poorly understood. Here the most densely sampled phylogenetic analysis of Arecoideae available to date is presented. The results are used to test the current classification of the subfamily and to identify priority areas for future research.

Methods

DNA sequence data for the low-copy nuclear genes PRK and RPB2 were collected from 190 palm species, covering 103 (96 %) genera of Arecoideae. The data were analysed using the parsimony ratchet, maximum likelihood, and both likelihood and parsimony bootstrapping.

Key Results and Conclusions

Despite the recovery of paralogues and pseudogenes in a small number of taxa, PRK and RPB2 were both highly informative, producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses. Simultaneous analyses of the combined data sets provided additional resolution and support. Two areas of incongruence between PRK and RPB2 were strongly supported by the bootstrap relating to the placement of tribes Chamaedoreeae, Iriarteeae and Reinhardtieae; the causes of this incongruence remain uncertain. The current classification within Arecoideae was strongly supported by the present data. Of the 14 tribes and 14 sub-tribes in the classification, only five sub-tribes from tribe Areceae (Basseliniinae, Linospadicinae, Oncospermatinae, Rhopalostylidinae and Verschaffeltiinae) failed to receive support. Three major higher level clades were strongly supported: (1) the RRC clade (Roystoneeae, Reinhardtieae and Cocoseae), (2) the POS clade (Podococceae, Oranieae and Sclerospermeae) and (3) the core arecoid clade (Areceae, Euterpeae, Geonomateae, Leopoldinieae, Manicarieae and Pelagodoxeae). However, new data sources are required to elucidate ambiguities that remain in phylogenetic relationships among and within the major groups of Arecoideae, as well as within the Areceae, the largest tribe in the palm family.     

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae

Background

Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity.

Results

We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99 % sequence identity in rDNA sequence and orthology across 85.6 % of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8 %) were present in Serratia while 33 (84.6 %) and 35 (89 %) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively.

Conclusion

The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result – killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1697-8) contains supplementary material, which is available to authorized users.     

Genome-wide profiling of in vivo LPS-responsive genes in splenic myeloid cells

Lipopolysaccharide (LPS), the major causative agent of bacterial sepsis, has been used by many laboratories in genome-wide expression profiling of the LPS response. However, these studies have predominantly used in vitro cultured macrophages (Macs), which may not accurately reflect the LPS response of these innate immune cells in vivo. To overcome this limitation and to identify inflammatory genes in vivo, we have profiled genome-wide expression patterns in non-lymphoid, splenic myeloid cells extracted directly from LPS-treated mice. Genes encoding factors known to be involved in mediating or regulating inflammatory processes, such as cytokines and chemokines, as well as many genes whose immunological functions are not well known, were strongly induced by LPS after 3 h or 8 h of treatment. Most of the highly LPSresponsive genes that we randomly selected from the microarray data were independently confirmed by quantitative RT-PCR, implying that our microarray data are quite reliable. When our in vivo data were compared to previously reported microarray data for in vitro LPS-treated Macs, a significant proportion (～20%) of the in vivo LPS-responsive genes defined in this study were specific to cells exposed to LPS in vivo, but a larger proportion of them (～60%) were influenced by LPS in both in vitro and in vivo settings. This result indicates that our in vivo LPS-responsive gene set includes not only previously identified in vitro LPS-responsive genes but also novel LPS-responsive genes. Both types of genes would be a valuable resource in the future for understanding inflammatory responses in vivo.