Nonfunctional Pseudogene Research Articles

Evolutionary dynamics of pro-inflammatory caspases in primates and rodents.

Caspase-1 and related proteases are key players in inflammation and innate immunity. Here, we characterize the evolutionary history of caspase-1 and its close relatives across 19 primates and 21 rodents, focusing on differences that may cause discrepancies between humans and animal studies. While caspase-1 has been retained in all these taxa, other members of the caspase-1 subfamily (caspase-4, -5, -11, -12, and CARD16, 17, and 18) each have unique evolutionary trajectories. Caspase-4 is found across simian primates, whereas we identified multiple pseudogenization and gene loss events in caspase-5, caspase-11, and the CARDs. Because caspases-4 and -11 are both key players in the non-canonical inflammasome pathway, we expected that these proteins would be likely to evolve rapidly. Instead, we found that these two proteins are largely conserved, whereas caspase-4's close paralog, caspase-5, showed significant indications of positive selection, as did primate caspase-1. Caspase-12 is a non-functional pseudogene in humans. We find this extends across most primates, although many rodents and some primates retain an intact, and likely functional, caspase-12. In mouse laboratory lines, we found that 50% of common strains carry non-synonymous variants that may impact the functions of caspase-11 and -12, and therefore recommend specific strains to be used (and avoided). Finally, unlike rodents, primate caspases have undergone repeated rounds of gene conversion, duplication, and loss leading to a highly dynamic pro-inflammatory caspase repertoire. Thus we uncovered many differences in the evolution of primate and rodent pro-inflammatory caspases, and discuss the potential implications of this history for caspase gene functions.

Chromosome-level genome assembly of the glass catfish ( Kryptopterus vitreolus) reveals molecular clues to its transparent phenotype.

Glass catfish ( Kryptopterus vitreolus) are notable in the aquarium trade for their highly transparent body pattern. This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in the main body, although certain black and silver pigments remain in the face and head. To date, however, the molecular mechanisms underlying this transparent phenotype remain largely unknown. To explore the genetic basis of this transparency, we constructed a chromosome-level haplotypic genome assembly for the glass catfish, encompassing 32 chromosomes and 23 344 protein-coding genes, using PacBio and Hi-C sequencing technologies and standard assembly and annotation pipelines. Analysis revealed a premature stop codon in the putative albinism-related tyrp1b gene, encoding tyrosinase-related protein 1, rendering it a nonfunctional pseudogene. Notably, a synteny comparison with over 30 other fish species identified the loss of the endothelin-3 ( edn3b) gene in the glass catfish genome. To investigate the role of edn3b, we generated edn3b -/- mutant zebrafish, which exhibited a remarkable reduction in black pigments in body surface stripes compared to wild-type zebrafish. These findings indicate that edn3b loss contributes to the transparent phenotype of the glass catfish. Our high-quality chromosome-scale genome assembly and identification of key genes provide important molecular insights into the transparent phenotype of glass catfish. These findings not only enhance our understanding of the molecular mechanisms underlying transparency in glass catfish, but also offer a valuable genetic resource for further research on pigmentation in various animal species.

Basidiomycete non-reducing polyketide synthases function independently of SAT domains

BackgroundNon-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary diversification into eleven clades further underscores the relevance of these multi-domain enzymes. Following current knowledge, NR-PKSs initiate polyketide assembly by an N-terminal starter unit:acyl transferase (SAT) domain that catalyzes the transfer of an acetyl starter from the acetyl-CoA thioester onto the acyl carrier protein (ACP).ResultsA comprehensive phylogenetic analysis of NR-PKSs established a twelfth clade from which three representatives, enzymes CrPKS1-3 of the webcap mushroom Cortinarius rufoolivaceus, were biochemically characterized. These basidiomycete synthases lack a SAT domain yet are fully functional hepta- and octaketide synthases in vivo. Three members of the other clade of basidiomycete NR-PKSs (clade VIII) were produced as SAT-domainless versions and analyzed in vivo and in vitro. They retained full activity, thus corroborating the notion that the SAT domain is dispensable for many basidiomycete NR-PKSs. For comparison, the ascomycete octaketide synthase atrochrysone carboxylic acid synthase (ACAS) was produced as a SAT-domainless enzyme as well, but turned out completely inactive. However, a literature survey revealed that some NR-PKSs of ascomycetes carry mutations within the catalytic motif of the SAT domain. In these cases, the role of the domain and the origin of the formal acetate unit remains open.ConclusionsThe role of SAT domains differs between asco- and basidiomycete NR-PKSs. For the latter, it is not part of the minimal set of NR-PKS domains and not required for function. This knowledge may help engineer compact NR-PKSs for more resource-efficient routes. From the genomic standpoint, seemingly incomplete or corrupted genes encoding SAT-domainless NR-PKSs should not automatically be dismissed as non-functional pseudogenes, but considered during genome analysis to decipher the potential arsenal of natural products of a given fungus.

"In the light of evolution:" keratins as exceptional tumor biomarkers.

Keratins (KRTs) are the intermediate filament-forming proteins of epithelial cells, classified, according to their physicochemical properties, into "soft" and "hard" keratins. They have a key role in several aspects of cancer pathophysiology, including cancer cell invasion and metastasis, and several members of the KRT family serve as diagnostic or prognostic markers. The human genome contains both, functional KRT genes and non-functional KRT pseudogenes, arranged in two uninterrupted clusters on chromosomes 12 and 17. This characteristic renders KRTs ideal for evolutionary studies. Herein, comprehensive phylogenetic analyses of KRT homologous proteins in the genomes of major taxonomic divisions were performed, so as to fill a gap in knowledge regarding the functional implications of keratins in cancer biology among tumor-bearing species. The differential expression profiles of KRTs in diverse types of cancers were investigated by analyzing high-throughput data, as well. Several KRT genes, including the phylogenetically conserved ones, were found to be deregulated across several cancer types and to participate in a common protein-protein interaction network. This indicates that, at least in cancer-bearing species, these genes might have been under similar evolutionary pressure, perhaps to support the same important function(s). In addition, semantic relations between KRTs and cancer were detected through extensive text mining. Therefore, by applying an integrative in silico pipeline, the evolutionary history of KRTs was reconstructed in the context of cancer, and the potential of using non-mammalian species as model organisms in functional studies on human cancer-associated KRT genes was uncovered.

Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome.

Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

A Review of the Important Role of CYP2D6 in Pharmacogenomics.

Cytochrome P450 2D6 (CYP2D6) is a critical pharmacogene involved in the metabolism of ~20% of commonly used drugs across a broad spectrum of medical disciplines including psychiatry, pain management, oncology and cardiology. Nevertheless, CYP2D6 is highly polymorphic with single-nucleotide polymorphisms, small insertions/deletions and larger structural variants including multiplications, deletions, tandem arrangements, and hybridisations with non-functional CYP2D7 pseudogenes. The frequency of these variants differs across populations, and they significantly influence the drug-metabolising enzymatic function of CYP2D6. Importantly, altered CYP2D6 function has been associated with both adverse drug reactions and reduced drug efficacy, and there is growing recognition of the clinical and economic burdens associated with suboptimal drug utilisation. To date, pharmacogenomic clinical guidelines for at least 48 CYP2D6-substrate drugs have been developed by prominent pharmacogenomics societies, which contain therapeutic recommendations based on CYP2D6-predicted categories of metaboliser phenotype. Novel algorithms to interpret CYP2D6 function from sequencing data that consider structural variants, and machine learning approaches to characterise the functional impact of novel variants, are being developed. However, CYP2D6 genotyping is yet to be implemented broadly into clinical practice, and so further effort and initiatives are required to overcome the implementation challenges and deliver the potential benefits to the bedside.

MutS homologue 4 and MutS homologue 5 Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization.

Crossovers (COs) ensure accurate chromosome segregation during meiosis while creating novel allelic combinations. Here, we show that allotetraploid (AABB) durum wheat (Triticum turgidum ssp. durum) utilizes two pathways of meiotic recombination. The class I pathway requires MSH4 and MSH5 (MutSγ) to maintain the obligate CO/chiasma and accounts for ∼85% of meiotic COs, whereas the residual ∼15% are consistent with the class II CO pathway. Class I and class II chiasmata are skewed toward the chromosome ends, but class II chiasmata are significantly more distal than class I chiasmata. Chiasma distribution does not reflect the abundance of double-strand breaks, detected by proxy as RAD51 foci at leptotene, but only ∼2.3% of these sites mature into chiasmata. MutSγ maintains the obligate chiasma despite a 5.4-kb deletion in MSH5B rendering it nonfunctional, which occurred early in the evolution of tetraploid wheat and was then domesticated into hexaploid (AABBDD) common wheat (Triticum aestivum), as well as an 8-kb deletion in MSH4D in hexaploid wheat, predicted to create a nonfunctional pseudogene. Stepwise loss of MSH5B and MSH4D following hybridization and whole-genome duplication may have occurred due to gene redundancy (as functional copies of MSH5A, MSH4A, and MSH4B are still present in the tetraploid and MSH5A, MSH5D, MSH4A, and MSH4B are present in the hexaploid) or as an adaptation to modulate recombination in allopolyploid wheat.

Identification of sorghum grain mold resistance loci through genome wide association mapping

Grain mold, caused by a consortium of pathogenic fungal species, is the most important disease of sorghum [Sorghum bicolor (L.) Moench]. Genome wide association study on 1425 diverse Ethiopian sorghum landraces identified a major grain mold resistance locus containing tightly linked and sequence related MYB transcription factor genes. The locus contains YELLOW SEED1 (Y1), a likely non-functional pseudo gene (Y2), and YELLOW SEED3 (Y3). SNPs and other sequence polymorphisms that alter the Y1 and Y3 genes correlated with susceptibility to grain mold and provided a strong genetic evidence. Accordingly, the expression of both Y1 and Y3 genes in the developing grain and glumes of a widely known susceptible sorghum line, RTx430, were severely reduced but significantly increased in the resistant line, RTx2911. In addition, the expression of flavonoid biosynthesis genes such as DIHYDROFLAVONOL 4-REDUCTASE 3 (DFR3) was significantly induced in the resistant line in response to inoculation by a mixture of spores from different molding fungi while the susceptible line displayed reduced expression. The data suggest that the MYB genes and their grain and glume specific expressions may determine the differential regulation of the flavonoid biosynthesis pathway genes, the synthesis of 3-Deoxyanthocynidins and ultimately responses to molding fungi. The study also suggested that resistance to grain mold may be negatively associated with grain functionality traits such as ‘injera’ making quality of sorghum.

Structure and function of L-threonine-3-dehydrogenase from the parasitic protozoan Trypanosoma brucei revealed by X-ray crystallography and geometric simulations.

Two of the world's most neglected tropical diseases, human African trypanosomiasis (HAT) and Chagas disease, are caused by protozoan parasites of the genus Trypanosoma. These organisms possess specialized metabolic pathways, frequently distinct from those in humans, which have potential to be exploited as novel drug targets. This study elucidates the structure and function of L-threonine-3-dehydrogenase (TDH) from T. brucei, the causative pathogen of HAT. TDH is a key enzyme in the metabolism of L-threonine, and an inhibitor of TDH has been shown to have trypanocidal activity in the procyclic form of T. brucei. TDH is a nonfunctional pseudogene in humans, suggesting that it may be possible to rationally design safe and specific therapies for trypanosomiasis by targeting this parasite enzyme. As an initial step, the TDH gene from T. brucei was expressed and the three-dimensional structure of the enzyme was solved by X-ray crystallography. In multiple crystallographic structures, T. brucei TDH is revealed to be a dimeric short-chain dehydrogenase that displays a considerable degree of conformational variation in its ligand-binding regions. Geometric simulations of the structure have provided insight into the dynamic behaviour of this enzyme. Furthermore, structures of TDH bound to its natural substrates and known inhibitors have been determined, giving an indication of the mechanism of catalysis of the enzyme. Collectively, these results provide vital details for future drug design to target TDH or related enzymes.

Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype.

The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in Arabidopsis thaliana. Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that AtSUC6Col-0 is a high-affinity H+-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of AtSUC6Col-0 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of AtSUC6 did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7Col-0 is regulated by intragenic regions. Transport activity of AtSUC7Col-0 could not be analyzed in baker’s yeast or Xenopus oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters in planta was developed. Plasma membrane localized SUCs including AtSUC6Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7Col-0 might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an in planta expression system.

Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin.

Copy number variants (CNVs) can substantially contribute to the pathogenic variant spectrum in several disease genes. The detection of this type of variant is complicated in genes with high homology to other genomic sequences, yet such genomics regions are more likely to lead to CNVs, making it critical to address detection in these settings. We developed a copy number analysis approach for high homology genes/regions that consisted of next-generation sequencing (NGS)-based dosage analysis accompanied by allele-specific droplet digital PCR (ddPCR) confirmatory testing. We applied this approach to copy number analysis in STRC, a gene with 98.9% homology to a nonfunctional pseudogene, pSTRC, and characterized its accuracy in detecting different copy number states by use of known samples. Using a cohort of 517 patients with hearing loss, we prospectively demonstrated the clinical utility of the approach, which contributed 30 of the 122 total positives (6%) to the diagnostic yield, increasing the overall yield from 17.6% to 23.6%. Positive STRC genotypes included homozygous (n = 15) or compound heterozygous (n = 8) deletions, or heterozygous deletions in trans with pathogenic sequence variants (n = 7). Finally, this approach limited ddPCR testing to cases with NGS copy number findings, thus markedly reducing the number of costly and laborious, albeit specific, ddPCR tests. NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues.

Whole-Genome Sequence of the Novel Enterobacter Bacteriophage Arya with an Integrase Pseudogene, Isolated from the Gut of the Formosan Subterranean Termite.

ABSTRACTWe isolated and sequenced the novel Enterobacter bacteriophage Arya from termite gut. The genome showed synteny to lytic bacteriophage genomes; however, the genome encodes a truncated, putatively nonfunctional integrase pseudogene. Lysogeny-related genes were previously observed in certain lytic phages, but their role and evolution remain unclear.

Identification of an archaeal maltooligosyltrehalose trehalohydrolase encoded by an interrupted gene.

Complete genome analysis of the thermoacidophilic Archaeon Sulfolobus tokodaii strain 7 revealed two open reading frames (ORF), namely, ST0926 and ST0927. These ORFs are interrupted by two putative insertions and encode for the N- and C-terminal fragments, respectively, of a putative Sulfolobus sp. maltooligosyltrehalose trehalohydrolase (StMTHase). Two specific deletion mutations, designed on the basis of sequence alignments of the adjacent ORFs and the published Sulfolobus sp. MTHases, allowed soluble expression in Escherichia coli of an active acidic and thermophilic enzyme. The purified enzyme exhibited a maximum amylolytic activity at 70 °C and pH 5.0, which resembled the optimal conditions of the Sulfolobus homologs. Furthermore, we report that these ORFs are actively co-transcribed in vivo, and we confirm the presence of insertions between them at the cDNA level. However, immunization and western blot experiments demonstrated no expression of ST0926 or the putative full-length StMTHase in vivo, indicating that they might exist as nonfunctional pseudogenes.

Red Algal Mitochondrial Genomes Are More Complete than Previously Reported.

The enslavement of an alpha-proteobacterial endosymbiont by the last common eukaryotic ancestor resulted in large-scale gene transfer of endosymbiont genes to the host nucleus as the endosymbiont transitioned into the mitochondrion. Mitochondrial genomes have experienced widespread gene loss and genome reduction within eukaryotes and DNA sequencing has revealed that most of these gene losses occurred early in eukaryotic lineage diversification. On a broad scale, more recent modifications to organelle genomes appear to be conserved and phylogenetically informative. The first red algal mitochondrial genome was sequenced more than 20 years ago, and an additional 29 Florideophyceae mitochondria have been added over the past decade. A total of 32 genes have been described to have been missing or considered non-functional pseudogenes from these Florideophyceae mitochondria. These losses have been attributed to endosymbiotic gene transfer or the evolution of a parasitic life strategy. Here we sequenced the mitochondrial genomes from the red algal parasite Choreocolax polysiphoniae and its host Vertebrata lanosa and found them to be complete and conserved in structure with other Florideophyceae mitochondria. This result led us to resequence the previously published parasite Gracilariophila oryzoides and its host Gracilariopsis andersonii, as well as reevaluate reported gene losses from published Florideophyceae mitochondria. Multiple independent losses of rpl20 and a single loss of rps11 can be verified. However by reannotating published data and resequencing specimens when possible, we were able to identify the majority of genes that have been reported as lost or pseudogenes from Florideophyceae mitochondria.

Molecular characterization and evolutionary origins of farinin genes in Brachypodium distachyon L.

Farinins are one of the oldest members of the gluten family in wheat and Aegilops species, and they influence dough properties. Here, we performed the first detailed molecular genetic study on farinin genes in Brachypodium distachyon L., the model species for Triticum aestivum. A total of 51 b-type farinin genes were cloned and characterized, including 27 functional and 24 non-functional pseudogenes from 14 different B. distachyon accessions. All genes were highly similar to those previously reported from wheat and Aegilops species. The identification of deduced amino acid sequences showed that b-type farinins across Triticeae genomes could be classified as b1-, b2-, b3-, and b4-type farinins; however, B. distachyon had only b3- and b4-type farinins. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that farinin genes are transcribed into mRNA in B. distachyon at much lower levels than in Triticeae, despite the presence of cis-acting elements in promoter regions. Phylogenetic analysis suggested that Brachypodium farinins may have closer relationships with common wheat and further confirmed four different types of b-type farinins in Triticeae and Brachypodium genomes, corresponding to b1, b2, b3 (group 1), and b4 (group 2). A putative evolutionary origin model of farinin genes in Brachypodium, Triticum, and the related species suggests that all b-type farinins diverged from their common ancestor ~3.2 million years ago (MYA). The b3 and b4 types could be considered older in the farinin family. The results explain the loss of b1- and b2-type farinin alleles in Brachypodium.

Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.

GeneScissors: a comprehensive approach to detecting and correcting spurious transcriptome inference owing to RNA-seq reads misalignment

Motivation: RNA-seq techniques provide an unparalleled means for exploring a transcriptome with deep coverage and base pair level resolution. Various analysis tools have been developed to align and assemble RNA-seq data, such as the widely used TopHat/Cufflinks pipeline. A common observation is that a sizable fraction of the fragments/reads align to multiple locations of the genome. These multiple alignments pose substantial challenges to existing RNA-seq analysis tools. Inappropriate treatment may result in reporting spurious expressed genes (false positives) and missing the real expressed genes (false negatives). Such errors impact the subsequent analysis, such as differential expression analysis. In our study, we observe that ∼3.5% of transcripts reported by TopHat/Cufflinks pipeline correspond to annotated nonfunctional pseudogenes. Moreover, ∼10.0% of reported transcripts are not annotated in the Ensembl database. These genes could be either novel expressed genes or false discoveries.Results: We examine the underlying genomic features that lead to multiple alignments and investigate how they generate systematic errors in RNA-seq analysis. We develop a general tool, GeneScissors, which exploits machine learning techniques guided by biological knowledge to detect and correct spurious transcriptome inference by existing RNA-seq analysis methods. In our simulated study, GeneScissors can predict spurious transcriptome calls owing to misalignment with an accuracy close to 90%. It provides substantial improvement over the widely used TopHat/Cufflinks or MapSplice/Cufflinks pipelines in both precision and F-measurement. On real data, GeneScissors reports 53.6% less pseudogenes and 0.97% more expressed and annotated transcripts, when compared with the TopHat/Cufflinks pipeline. In addition, among the 10.0% unannotated transcripts reported by TopHat/Cufflinks, GeneScissors finds that >16.3% of them are false positives.Availability: The software can be downloaded at http://csbio.unc.edu/genescissors/Contact: weiwang@cs.ucla.eduSupplementary information: Supplementary data are available at Bioinformatics online.

High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6.

High-resolution melt (HRM) analysis using 'release-on-demand' dyes, such as EvaGreen(®) has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African-Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.

Intragenomic sequence variation at the ITS1–ITS2 region and at the 18S and 28S nuclear ribosomal DNA genes of the New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae: Mollusca)

Molecular genetic analysis was conducted on two populations of the invasive non-native New Zealand mud snail (Potamopyrgus antipodarum), one from a freshwater ecosystem in Devil’s Lake (Oregon, USA) and the other from an ecosystem of higher salinity in the Columbia River estuary (Hammond Harbor, Oregon, USA). To elucidate potential genetic differences between the two populations, three segments of nuclear ribosomal DNA (rDNA), the ITS1-ITS2 regions and the 18S and 28S rDNA genes were cloned and sequenced. Variant sequences within each individual were found in all three rDNA segments. Folding models were utilized for secondary structure analysis and results indicated that there were many sequences which contained structure-altering polymorphisms, which suggests they could be nonfunctional pseudogenes. In addition, analysis of molecular variance (AMOVA) was used for hierarchical analysis of genetic variance to estimate variation within and among populations and within individuals. AMOVA revealed significant variation in the ITS region between the populations and among clones within individuals, while in the 5.8S rDNA significant variation was revealed among individuals within the two populations. High levels of intragenomic variation were found in the ITS regions, which are known to be highly variable in many organisms. More interestingly, intragenomic variation was also found in the 18S and 28S rDNA, which has rarely been observed in animals and is so far unreported in Mollusca. We postulate that in these P. antipodarum populations the effects of concerted evolution are diminished due to the fact that not all of the rDNA genes in their polyploid genome should be essential for sustaining cellular function. This could lead to a lessening of selection pressures, allowing mutations to accumulate in some copies, changing them into variant sequences.