Pseudogene Sequences Research Articles

The physical and evolutionary energy landscapes of devolved protein sequences corresponding to pseudogenes

Protein evolution is guided by structural, functional, and dynamical constraints ensuring organismal viability. Pseudogenes are genomic sequences identified in many eukaryotes that lack translational activity due to sequence degradation and thus over time have undergone "devolution." Previously pseudogenized genes sometimes regain their protein-coding function, suggesting they may still encode robust folding energy landscapes despite multiple mutations. We study both the physical folding landscapes of protein sequences corresponding to human pseudogenes using the Associative Memory, Water Mediated, Structure and Energy Model, and the evolutionary energy landscapes obtained using direct coupling analysis (DCA) on their parent protein families. We found that generally mutations that have occurred in pseudogene sequences have disrupted their native global network of stabilizing residue interactions, making it harder for them to fold if they were translated. In some cases, however, energetic frustration has apparently decreased when the functional constraints were removed. We analyzed this unexpected situation for Cyclophilin A, Profilin-1, and Small Ubiquitin-like Modifier 2 Protein. Our analysis reveals that when such mutations in the pseudogene ultimately stabilize folding, at the same time, they likely alter the pseudogenes' former biological activity, as estimated by DCA. We localize most of these stabilizing mutations generally to normally frustrated regions required for binding to other partners.

Four classic "de novo" genes all have plausible homologs and likely evolved from retro-duplicated or pseudogenic sequences.

Despite being previously regarded as extremely unlikely, the idea that entirely novel protein-coding genes can emerge from non-coding sequences has gradually become accepted over the past two decades. Examples of "de novo origination", resulting in lineage-specific "orphan" genes, lacking coding orthologs, are now produced every year. However, many are likely cases of duplicates that are difficult to recognize. Here, I re-examine the claims and show that four very well-known examples of genes alleged to have emerged completely "from scratch"- FLJ33706 in humans, Goddard in fruit flies, BSC4 in baker's yeast and AFGP2 in codfish-may have plausible evolutionary ancestors in pre-existing genes. The first two are likely highly diverged retrogenes coding for regulatory proteins that have been misidentified as orphans. The antifreeze glycoprotein, moreover, may not have evolved from repetitive non-genic sequences but, as in several other related cases, from an apolipoprotein that could have become pseudogenized before later being reactivated. These findings detract from various claims made about de novo gene birth and show there has been a tendency not to invest the necessary effort in searching for homologs outside of a very limited syntenic or phylostratigraphic methodology. A robust approach is used for improving detection that draws upon similarities, not just in terms of statistical sequence analysis, but also relating to biochemistry and function, to obviate notable failures to identify homologs.

Uncovering a 500 million year old history and evidence of pseudogenization for TLR15.

Toll like receptors (TLRs) are at the front line of pathogen recognition and host immune response. Many TLR genes have been described to date with some being found across metazoans while others are restricted to specific lineages. A cryptic member of the TLR gene family, TLR15, has a unique phylogenetic distribution. Initially described in extant species of birds and reptiles, an ortholog has been reported for cartilaginous fish. Here, we significantly expanded the evolutionary analysis of TLR15 gene evolution, taking advantage of large genomic and transcriptomic resources available from different lineages of vertebrates. Additionally, we objectively search for TLR15 in lobe-finned and ray-finned fish, as well as in cartilaginous fish and jawless vertebrates. We confirm the presence of TLR15 in early branching jawed vertebrates - the cartilaginous fish, as well as in basal Sarcopterygii - in lungfish. However, within cartilaginous fish, the gene is present in Holocephalans (all three families) but not in Elasmobranchs (its sister-lineage). Holocephalans have long TLR15 protein sequences that disrupt the typical TLR structure, and some species display a pseudogene sequence due to the presence of frameshift mutations and early stop codons. Additionally, TLR15 has low expression levels in holocephalans when compared with other TLR genes. In turn, lungfish also have long TLR15 protein sequences but the protein structure is not compromised. Finally, TLR15 presents several sites under negative selection. Overall, these results suggest that TLR15 is an ancient TLR gene and is experiencing ongoing pseudogenization in early-branching vertebrates.

Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

ABSTRACT While DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterized the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole-genome bisulphite sequencing (WGBS) and bisulphite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723 ± 7,711) and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49 ± 0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.

Processed pseudogenes: A substrate for evolutionary innovation: Retrotransposition contributes to genome evolution by propagating pseudogene sequences with rich regulatory potential throughout the genome.

Processed pseudogenes may serve as a genetic reservoir for evolutionary innovation. Here, we argue that through the activity of long interspersed element-1 retrotransposons, processed pseudogenes disperse coding and noncoding sequences rich with regulatory potential throughout the human genome. While these sequences may appear to be non-functional, a lack of contemporary function does not prohibit future development of biological activity. Here, we discuss the dynamic evolution of certain processed pseudogenes into coding and noncoding genes and regulatory elements, and their implication in wide-ranging biological and pathological processes. Also see the video abstract here: https://youtu.be/iUY_mteVoPI.

Comparative analysis of zooplankton diversities and compositions estimated from complement DNA and genomic DNA amplicons, metatranscriptomics, and morphological identifications

AbstractCommunity-based diversity analyses, such as metabarcoding, are increasingly popular in the field of metazoan zooplankton community ecology. However, some of the methodological uncertainties remain, such as the potential inflation of diversity estimates resulting from contamination by pseudogene sequences. Furthermore, primer affinity to specific taxonomic groups might skew community composition and structure during PCR. In this study, we estimated OTU (operational taxonomic unit) richness, Shannon’s H’, and the phylum-level community composition of samples from a coastal zooplankton community using four approaches: complement DNA (cDNA) and genomic DNA (gDNA) mitochondrial COI (Cytochrome oxidase subunit I) gene amplicon, metatranscriptome sequencing, and morphological identification. Results of mismatch distribution demonstrated that 90% is good threshold percentage to differentiate intra- and inter-species. Moderate level of correlations appeared upon comparing the species/OTU richness estimated from the different methods. Results strongly indicated that diversity inflation occurred in the samples amplified from gDNA because of mitochondrial pseudogene contamination (overall, gDNA produced two times more richness compared with cDNA amplicons). The unique community compositions observed in the PCR-based methods indicated that taxonomic amplification bias had occurred during the PCR. Therefore, it is recommended that PCR-free approaches be used whenever resolving community structure represents an essential aspect of the analysis.

Profile hidden Markov model sequence analysis can help remove putative pseudogenes from DNA barcoding and metabarcoding datasets

BackgroundPseudogenes are non-functional copies of protein coding genes that typically follow a different molecular evolutionary path as compared to functional genes. The inclusion of pseudogene sequences in DNA barcoding and metabarcoding analysis can lead to misleading results. None of the most widely used bioinformatic pipelines used to process marker gene (metabarcode) high throughput sequencing data specifically accounts for the presence of pseudogenes in protein-coding marker genes. The purpose of this study is to develop a method to screen for nuclear mitochondrial DNA segments (nuMTs) in large COI datasets. We do this by: (1) describing gene and nuMT characteristics from an artificial COI barcode dataset, (2) show the impact of two different pseudogene removal methods on perturbed community datasets with simulated nuMTs, and (3) incorporate a pseudogene filtering step in a bioinformatic pipeline that can be used to process Illumina paired-end COI metabarcode sequences. Open reading frame length and sequence bit scores from hidden Markov model (HMM) profile analysis were used to detect pseudogenes.ResultsOur simulations showed that it was more difficult to identify nuMTs from shorter amplicon sequences such as those typically used in metabarcoding compared with full length DNA barcodes that are used in the construction of barcode libraries. It was also more difficult to identify nuMTs in datasets where there is a high percentage of nuMTs. Existing bioinformatic pipelines used to process metabarcode sequences already remove some nuMTs, especially in the rare sequence removal step, but the addition of a pseudogene filtering step can remove up to 5% of sequences even when other filtering steps are in place.ConclusionsOpen reading frame length filtering alone or combined with hidden Markov model profile analysis can be used to effectively screen out apparent pseudogenes from large datasets. There is more to learn from COI nuMTs such as their frequency in DNA barcoding and metabarcoding studies, their taxonomic distribution, and evolution. Thus, we encourage the submission of verified COI nuMTs to public databases to facilitate future studies.

Dealing with Pseudogenes in Molecular Diagnostics in the Next Generation Sequencing Era.

Presence of pseudogenes is a dreadful issue in next generation sequencing (NGS), because their contamination can interfere with the detection of variants in the genuine gene and generate false positive and false negative variants.In this chapter we focus on issues related to the application of NGS strategies for analysis of genes with pseudogenes in a clinical setting. The degree to which a pseudogene impacts the ability to accurately detect and map variants in its parent gene depends on the degree of similarity (homology) with the parent gene itself. Hereby, target enrichment and mapping strategies are crucial factors to avoid "contaminating" pseudogene sequences. For target enrichment, we describe advantages and disadvantages of PCR- and capture-based strategies. For mapping strategies, we discuss crucial parameters that need to be considered to accurately distinguish sequences of functional genes from pseudogenic sequences. Finally, we discuss some examples of genes associated with Mendelian disorders, for which interesting NGS approaches are described to avoid interference with pseudogene sequences.

CRISPR/Cas Technologies Applied to Pseudogenes.

Pseudogenes have been considered as nonfunctional copies of their parental genes for a long time. Indeed, they have been often defined "junk DNA" or "transcriptional noise." However, with the identification of their involvement in several biological processes, the necessity of their study is inevitably growing up. The manipulation of pseudogene expression is complicated by their high homology with parental genes and bythe fact that most of them work at the transcriptional level as noncoding RNAs. With the advent of CRISPR/Cas technology, these problems can be overcome. Particularly, as we describe in this chapter, it is possible: To perform genome editing, obtaining the complete elimination of the pseudogene genomic sequence (knock-out), preventing pseudogene transcription, introducing specific mutations in the pseudogene sequence, or introducing a specific sequence (knock-in). To positively or negatively manipulate pseudogene transcription. To target pseudogene RNA and negatively regulate its expression. To edit pseudogene DNA and RNA and alter a specific sequence. Moreover, CRISPR/Cas technology can be used as an RNA Binding Protein system for molecular biology techniques (such as RNA immunoprecipitation and pull-down), as well as for transcript tracking and live imaging.

Identification of Mitochondrial DNA (NUMTs) in the Nuclear Genome of Daphnia magna.

This is the first study in which the Daphnia magna (D. magna) nuclear genome (nDNA) obtained from the GenBank database was analyzed for pseudogene sequences of mitochondrial origin. To date, there is no information about pseudogenes localized in D. magna genome. This study aimed to identify NUMTs, their length, homology, and location for potential use in evolutionary studies and to check whether their occurrence causes co-amplification during mitochondrial genome (mtDNA) analyses. Bioinformatic analysis showed 1909 fragments of the mtDNA of D. magna, of which 1630 were located in ten linkage groups (LG) of the nDNA. The best-matched NUMTs covering >90% of the gene sequence have been identified for two mt-tRNA genes, and they may be functional nuclear RNA molecules. Isolating the total DNA in mtDNA studies, co-amplification of nDNA fragments is unlikely in the case of amplification of the whole tRNA genes as well as fragments of other genes. It was observed that TRNA-MET fragments had the highest level of sequence homology, thus they could be evolutionarily the youngest. The lowest homology was found in the D-loop-derived pseudogene. It may probably be the oldest NUMT incorporated into the nDNA; however, further analysis is necessary.

Evolution of major histocompatibility complex class I genes in the sable Martes zibellina (Carnivora, Mustelidae).

The molecules encoded by major histocompatibility complex (MHC) genes play an essential role in the adaptive immune response among vertebrates. We investigated the molecular evolution of MHC class I genes in the sable Martes zibellina. We isolated 26 MHC class I sequences, including 12 putatively functional sequences and 14 pseudogene sequences, from 24 individuals from two geographic areas of northeast China. The number of putatively functional sequences found in a single individual ranged from one to five, which might be at least 1–3 loci. We found that both balancing selection and recombination contribute to evolution of MHC class I genes in M. zibellina. In addition, we identified a candidate nonclassical MHC class I lineage in Carnivora, which may have preceded the divergence (about 52–57 Mya) of Caniformia and Feliformia. This may contribute to further understanding of the origin and evolution of nonclassical MHC class I genes. Our study provides important immune information of MHC for M. zibellina, as well as other carnivores.

Genome-wide codon usage pattern analysis reveals the correlation between codon usage bias and gene expression in Cuscuta australis

The protein-coding genes and pseudogenes of Cuscuta australis had the diverse contribution to the formation and evolution of parasitism. The codon usage pattern analysis of these two type genes could be used to understand the gene transcription and translation. In this study, we systematically analyzed the codon usage patterns of protein-coding sequences and pseudogenes sequences in C. australis. The results showed that the high frequency codons of protein coding sequences and pseudogenes had the same A/U bias in the third position. However, these two sequences had converse bias at the third base in optimal codons: the protein coding sequences preferred G/C-ending codons while pseudogene sequences preferred A/U-ending codons. Neutrality plot and effective number of codons plot revealed that natural selection played a more important role than mutation pressure in two sequences codon usage bias. Furthermore, the gene expression level had a significant positive correlation with codon usage bias in C. australis. Highly-expressed protein coding genes exhibited a higher codon bias than lowly-expressed genes. Meanwhile, the high-expression genes tended to use G/C-ending synonymous codons. This result further verified the optimal codons usage bias and its correlation with the gene expression in C. australis.

Molecular phylogeny of mulberries reconstructed from ITS and two cpDNA sequences.

BackgroundSpecies in the genus Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information.MethodsWe sequenced the ITS regions and the chloroplast DNA regions TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry.ResultsWe found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades.ConclusionsTwo species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution.

Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking.

Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR–Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR–Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.

Convergent horizontal gene transfer and cross-talk of mobile nucleic acids in parasitic plants.

Horizontal gene transfer (HGT), the movement and genomic integration of DNA across species boundaries, is commonly associated with bacteria and other microorganisms, but functional HGT (fHGT) is increasingly being recognized in heterotrophic parasitic plants that obtain their nutrients and water from their host plants through direct haustorial feeding. Here, in the holoparasitic stem parasite Cuscuta, we identify 108 transcribed and probably functional HGT events in Cuscuta campestris and related species, plus 42 additional regions with host-derived transposon, pseudogene and non-coding sequences. Surprisingly, 18 Cuscuta fHGTs were acquired from the same gene families by independent HGT events in Orobanchaceae parasites, and the majority are highly expressed in the haustorial feeding structures in both lineages. Convergent retention and expression of HGT sequences suggests an adaptive role for specific additional genes in parasite biology. Between 16 and 20 of the transcribed HGT events are inferred as ancestral in Cuscuta based on transcriptome sequences from species across the phylogenetic range of the genus, implicating fHGT in the successful radiation of Cuscuta parasites. Genome sequencing of C. campestris supports transfer of genomic DNA-rather than retroprocessed RNA-as the mechanism of fHGT. Many of the C. campestris genes horizontally acquired are also frequent sources of 24-nucleotide small RNAs that are typically associated with RNA-directed DNA methylation. One HGT encoding a leucine-rich repeat protein kinase overlaps with a microRNA that has been shown to regulate host gene expression, suggesting that HGT-derived parasite small RNAs may function in the parasite-host interaction. This study enriches our understanding of HGT by describing a parasite-host system with unprecedented gene exchange that points to convergent evolution of HGT events and the functional importance of horizontally transferred coding and non-coding sequences.

Enhanced Production of Recombinant Alcohol Dehydrogenase Using the Genetically Engineered Escherichia coli Strain that Heterologously Expresses Carrot Heat Shock Protein 70.

Escherichia coli (E. coli) has been widely used as a host organism for producing recombinant proteins such as biocatalysts, antibody fragments, and therapeutic hormones. To enhance recombinant protein production, many E. coli strains have been genetically engineered on practical purposes. In this study, we developed the engineered E. coli strain expressing Heat shock protein 70, DcHsp70, from carrot (Daucus carota L.). The DNA construct for DcHsp70 expression, Lipoprotein promoter-DcHsp70 gene-Flippase recognition target cassette, which is flanked by the insertion site yddE pseudogene sequences, was generated by overlap PCR and inserted into the E. coli genome by lambda Red-mediated homologous recombination. To examine if the engineered E. coli cells can effectively produce recombinant proteins, the alcohol dehydrogenase (ADH) gene from a thermophile, Geobacillus stearothermophilus, was cloned into a pET11a expression vector and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. Compared to wild type, the genetically engineered E. coli expressing DcHsp70 exhibited up to approximately 11-fold higher production of his-tagged ADH, mostly in soluble forms. The his-ADH protein that was purified from the engineered cells exhibited the enzyme activity. The genetically engineered E. coli developed in this study can be useful for the efficient production of recombinant proteins, such as recombinant ADH.

A sensitive and accurate test for simultaneous detection of 36 BRAF and NRAS mutations.

e24153 Background: Activating mutations in the mitogen-activated protein kinase pathway are among the most common mutations found in cancer cells. Because of the interaction between BRAF and NRAS, there is interest in simultaneous testing for mutation status of these genes. We have developed a multiplexed, allele-specific PCR kit for broad and sensitive detection of 36 BRAF and NRAS mutations. The BRAF/NRAS Mutation Test kit has been verified for use with FFPE tissue and plasma (Research Use Only).The sample to result can be performed in eight hours or less. A web-based analysis tool enables automated and rapid interpretation of results. We describe the studies done to verify the test performance and to evaluate its correlation with an Illumina MiSeq method. Methods: We tested contrived specimens as well as FFPE tissue and plasma specimens from melanoma, non-small cell lung, and colorectal cancer patients. Results for analytical sensitivity, exclusivity, repeatability, and correlation with the MiSeq method are reported. Results: The BRAF/NRAS Mutation Test demonstrated high sensitivity, specificity and broad mutation coverage. Repeatability was 100% across all samples, operators, and instruments. Analytical sensitivity was high for tissue and plasma samples. 1% sensitivity was achieved for 34 mutations using mutant plasmid DNA in 50ng genomic DNA. 2.5% mutant allele frequency in 50ng FFPE DNA and as low as 0.03% from 2mL plasma were detected. Plasmids containing highly similar HRAS, KRAS, and KRAS pseudogene plus A-RAF, C-RAF, and BRAF pseudogene sequences were not detected, showing specificity for NRAS and BRAF. The test showed high concordance for 164 tumor FFPE tissue from melanoma, CRC, and NSCLC (98.2%) and 186 plasma specimens from melanoma and CRC (98.9%) when compared to MiSeq sequencing. Conclusions: The BRAF/NRAS Mutation Test is a sensitive and reproducible kit for the detection of 11 BRAF mutations in exons 11 and 15 and 25 NRAS mutations in exons 2, 3, and 4. The simplified lab workflow and assay reliability make it suitable for the accurate detection of mutations from tissue and plasma biopsies.

Chromosomal dynamics, molecular organization and evolutionary patterns of major ribosomal DNA in the Grasshopper Abracris flavolineata (Acrididae)

In the grasshopper Abracris flavolineata it was documented huge intrapopulation variation for 18S rDNA in individuals collected in six populations, but causes for this pattern was not understand yet. In this work we aimed to elucidate the molecular structure, evolutionary patterns of the rDNA 18S distribution and its causes in the genome of A. flavolineata. It was used a combination of classical cytogenetic techniques, mapping of18S rDNA through Fluorescence in situ hybridization (FISH) and genomics analyses from entire sequenced genome by Illumina hiseq 2000. It was analyzed 48 individuals collected in six distantly sampling places from Brazil and Argentina. FISH analyses confirmed the variability of 18S rDNA for all six populations that presented between 6-11 chomosomes labeled. Signals were observed in chromosomes 1, 3, 6 and 9 in all individuals and also it was noticed variation of cluster sizes. It was not found a fixed pattern at intra- or interpopulation levels in population from the same geographic region or between distant ones. Silver nitrate staning revealed Nocleolar Organizer Regions (NORs) in chromosome 9 of all individuals, indicating that the expression of the rDNA cluster in this chromosome could be under selection to be expressed, and also could represent ancestral placement of this sequence. It is a pattern also shared with congeneric species. In some individuals chromosome 10 presented NORs, however for some of them FISH signals in this bivalent was not observed, indicating cryptic loci in accordance with ‘dispersion-amplification-deletion’ model, suggested also for rDNA dispersion for other grasshoppers. Finally the reconstruction of entire 45S rDNA presented different coverages in the conserved genes (18S, 5.8S e 25S) and in the internal transcripts (ITS 1 e ITS 2) reveling that huge part of the copies are pseudogenes. The transposons Nimb and R2 are present in 45S rDNA, although apparently they are not able to transpose sequences. All data together suggests that the high amount of rDNA in A. flavolineata genome is caused by occurrence of pseudogene sequences that were moved random between chromosomes caused most parsimonialy by ectopic recombination, extra-chromosomal DNA or even by transposons not localized in the 45S cluster

Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels

Next-generation sequencing (NGS) technology has rapidly replaced Sanger sequencing in the assessment of sequence variations in clinical genetics laboratories. One major limitation of current NGS approaches is the ability to detect copy number variations (CNVs) approximately >50 bp. Because these represent a major mutational burden in many genetic disorders, parallel CNV assessment using alternate supplemental methods, along with the NGS analysis, is normally required, resulting in increased labor, costs, and turnaround times. The objective of this study was to clinically validate a novel CNV detection algorithm using targeted clinical NGS gene panel data. We have applied this approach in a retrospective cohort of 391 samples and a prospective cohort of 2375 samples and found a 100% sensitivity (95% CI, 89%-100%) for 37 unique events and a high degree of specificity to detect CNVs across nine distinct targeted NGS gene panels. This NGS CNV pipeline enables stand-alone first-tier assessment for CNV and sequence variants in a clinical laboratory setting, dispensing with the need for parallel CNV analysis using classic techniques, such as microarray, long-range PCR, or multiplex ligation-dependent probe amplification. This NGS CNV pipeline can also be applied to the assessment of complex genomic regions, including pseudogenic DNA sequences, such as the PMS2CL gene, and to mitochondrial genome heteroplasmy detection.

RRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data.

The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes.