Inversion variants in human and primate genomes.

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.

A Closed-Tube Nested Quantitative PCR Assay for Rapid Detection of Intron 22 Inversions in the Factor VIII Gene.

BACKGROUND: An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. METHODS: We report here a new method using a single closed-tube nested quantitative PCR (CN-qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD-PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD-PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. RESULTS: Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN-qPCR assay and the standard LD-PCR assay. CN-qPCR successfully made calls for all samples, whereas LD-PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN-qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. CONCLUSIONS: This new CN-qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.

Long-read sequencing for rare human genetic diseases.

During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.

Detailed analysis of inversions predicted between two human genomes: errors, real polymorphisms, and their origin and population distribution.

The growing catalogue of structural variants in humans often overlooks inversions as one of the most difficult types of variation to study, even though they affect phenotypic traits in diverse organisms. Here, we have analysed in detail 90 inversions predicted from the comparison of two independently assembled human genomes: the reference genome (NCBI36/HG18) and HuRef. Surprisingly, we found that two thirds of these predictions (62) represent errors either in assembly comparison or in one of the assemblies, including 27 misassembled regions in HG18. Next, we validated 22 of the remaining 28 potential polymorphic inversions using different PCR techniques and characterized their breakpoints and ancestral state. In addition, we determined experimentally the derived allele frequency in Europeans for 17 inversions (DAF = 0.01-0.80), as well as the distribution in 14 worldwide populations for 12 of them based on the 1000 Genomes Project data. Among the validated inversions, nine have inverted repeats (IRs) at their breakpoints, and two show nucleotide variation patterns consistent with a recurrent origin. Conversely, inversions without IRs have a unique origin and almost all of them show deletions or insertions at the breakpoints in the derived allele mediated by microhomology sequences, which highlights the importance of mechanisms like FoSTeS/MMBIR in the generation of complex rearrangements in the human genome. Finally, we found several inversions located within genes and at least one candidate to be positively selected in Africa. Thus, our study emphasizes the importance of careful analysis and validation of large-scale genomic predictions to extract reliable biological conclusions.

Complete plastome sequences of Picea asperata and P. crassifolia and comparative analyses with P. abies and P. morrisonicola.

Picea asperata and P. crassifolia have sympatric ranges and are closely related, but the differences between these species at the plastome level are unknown. To better understand the patterns of variation among Picea plastomes, the complete plastomes of P. asperata and P. crassifolia were sequenced. Then, the plastomes were compared with the complete plastomes of P. abies and P. morrisonicola, which are closely and distantly related to the focal species, respectively. We also used these sequences to construct phylogenetic trees to determine the relationships among and between the four species as well as additional taxa from Pinaceae and other gymnosperms. Analysis of our sequencing data allowed us to identify 438 single nucleotide polymorphism (SNPs) point mutation events, 95 indel events, four inversion events, and seven highly variable regions, including six gene spacer regions (psbJ-petA, trnT-psaM, trnS-trnD, trnL-rps4, psaC-ccsA, and rps7-trnL) and one gene (ycf1). The highly variable regions are appropriate targets for future use in the phylogenetic reconstructions of closely related, sympatric species of Picea as well as Pinaceae in general.

Targeted conservative formation of cointegrates between two DNA molecules containing IS26 occurs via strand exchange at either IS end.

We recently proposed a model for targeted, conservative cointegrate formation between DNA molecules each containing a copy of IS26, that involves Tnp26-catalyzed strand exchange occurring at either the two left ends or the two right ends of the IS. Here, this model was validated by altering the bases at the outer left terminus, right terminus or both termini of one IS26. The correct bases at both ends were required in the untargeted replicative mode. However, when only one end was altered in one participating IS the frequency of targeted, conservative cointegrate formation was not reduced. The distribution of the altered bases in the cointegrates confirmed that the reaction occurred at the end where the terminal bases of both IS were correct, and cointegrates were not formed when both ends of the same IS were altered. The terminal bases of the active IS26 were also required to support deletion of the aphA1a translocatable unit (TU) from Tn4352B. The choices made by an incoming TU with a wild-type IS26 when the target plasmid included one wild-type IS26 and one with a frameshift in tnp26 demonstrated that Tnp26 exhibits a strong preference for cis action.

Core-genome scaffold comparison reveals the prevalence that inversion events are associated with pairs of inverted repeats.

BACKGROUND: Genome rearrangement describes gross changes of chromosomal regions, plays an important role in evolutionary biology and has profound impacts on phenotype in organisms ranging from microbes to humans. With more and more complete genomes accomplished, lots of genomic comparisons have been conducted in order to find genome rearrangements and the mechanisms which underlie the rearrangement events. In our opinion, genomic comparison of different individuals/strains within the same species (pan-genome) is more helpful to reveal the mechanisms for genome rearrangements since genomes of the same species are much closer to each other. RESULTS: We study the mechanism for inversion events via core-genome scaffold comparison of different strains within the same species. We focus on two kinds of bacteria, Pseudomonas aeruginosa and Escherichia coli, and investigate the inversion events among different strains of the same species. We find an interesting phenomenon that long (larger than 10,000 bp) inversion regions are flanked by a pair of Inverted Repeats (IRs). This mechanism can also explain why the breakpoint reuses for inversion events happen. We study the prevalence of the phenomenon and find that it is a major mechanism for inversions. The other observation is that for different rearrangement events such as transposition and inverted block interchange, the two ends of the swapped regions are also associated with repeats so that after the rearrangement operations the two ends of the swapped regions remain unchanged. To our knowledge, this is the first time such a phenomenon is reported for transposition event. CONCLUSIONS: In both Pseudomonas aeruginosa and Escherichia coli strains, IRs were found at the two ends of long sequence inversions. The two ends of the inversion remained unchanged before and after the inversion event. The existence of IRs can explain the breakpoint reuse phenomenon. We also observed that other rearrangement operations such as transposition, inverted transposition, and inverted block interchange, had repeats (not necessarily inverted) at the ends of each segment, where the ends remained unchanged before and after the rearrangement operations. This suggests that the conservation of ends could possibly be a popular phenomenon in many types of chromosome rearrangement events.

Discovery of large genomic inversions using long range information.

BACKGROUND: Although many algorithms are now available that aim to characterize different classes of structural variation, discovery of balanced rearrangements such as inversions remains an open problem. This is mainly due to the fact that breakpoints of such events typically lie within segmental duplications or common repeats, which reduces the mappability of short reads. The algorithms developed within the 1000 Genomes Project to identify inversions are limited to relatively short inversions, and there are currently no available algorithms to discover large inversions using high throughput sequencing technologies. RESULTS: Here we propose a novel algorithm, VALOR, to discover large inversions using new sequencing methods that provide long range information such as 10X Genomics linked-read sequencing, pooled clone sequencing, or other similar technologies that we commonly refer to as long range sequencing. We demonstrate the utility of VALOR using both pooled clone sequencing and 10X Genomics linked-read sequencing generated from the genome of an individual from the HapMap project (NA12878). We also provide a comprehensive comparison of VALOR against several state-of-the-art structural variation discovery algorithms that use whole genome shotgun sequencing data. CONCLUSIONS: In this paper, we show that VALOR is able to accurately discover all previously identified and experimentally validated large inversions in the same genome with a low false discovery rate. Using VALOR, we also predicted a novel inversion, which we validated using fluorescent in situ hybridization. VALOR is available at https://github.com/BilkentCompGen/VALOR.

Conditionals by inversion provide a universal method for the generation of conditional alleles.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.

Genomic inversion caused by gamma irradiation contributes to downregulation of a WBC11 homolog in bloomless sorghum.

Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight-a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F2 bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.

A novel inverted 17p13.3 microduplication disrupting PAFAH1B1 (LIS1) in a girl with syndromic lissencephaly.

We describe a female patient with mild lissencephaly (pachygyria), severe intellectual disability, and facial dysmorphisms with an inverted 1.4 Mb microduplication of chromosome 17p13.3. The 17p13.3 microduplication syndrome is associated with mild intellectual disabiltiy and contains, among others, the PAFAH1B1 (LIS1) gene, whereas microdeletions of the same segment cause Miller-Dieker syndrome (MDS) with severe to profound retardation. The duplication identified in our patient encompasses 29 genes, including CRK and YWHAE. The proximal breakpoint of the duplication is located in the first intron of the PAFAH1B1 gene. Analysis of total RNA showed that only one PAFAH1B1 allele is expressed. Therefore, this patient has a unique alteration: a duplication including YWHAE and CRK and haploinsufficiency of PAFAH1B1. Overexpression of YWHAE is associated with macrosomia, mild developmental delay, autism and facial dysmorphisms, and deletion of PAFAH1B1 alone leads to isolated lissencephaly (ILS). The patient described here shares features with MDS, but she is affected to a lesser degree. Her facial features are similar to MDS, and she has manifestations seen in other cases with YWHAE duplication.

Child development and structural variation in the human genome.

Structural variation of the human genome sequence is the insertion, deletion, or rearrangement of stretches of DNA sequence sized from around 1,000 to millions of base pairs. Over the past few years, structural variation has been shown to be far more common in human genomes than previously thought. Very little is currently known about the effects of structural variation on normal child development, but such effects could be of considerable significance. This review provides an overview of the phenomenon of structural variation in the human genome sequence, describing the novel genomics technologies that are revolutionizing the way structural variation is studied and giving examples of genomic structural variations that affect child development.

Becker muscular dystrophy due to an inversion of exons 23 and 24 of the DMD gene.

The use of hybridization-based methods for Duchenne muscular dystrophy (DMD) mutation analysis is increasingly common. We report a case of Becker muscular dystrophy in which discrepant results between a polymerase chain reaction (PCR)-based single-condition amplification/internal primer (SCAIP) and a comparative genomic hybridization assay incompletely characterized the mutation (an inversion of exons 23 and 24). These results demonstrate the limits of sensitivity and specificity of both tests, and highlight the need for more detailed analysis when intronic deletions are detected by comparative genome hybridization methods.

[Localization of the Dras1 sequence to polytene chromosomes in sibling species of the Drosophila virilis group].

Drasl gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and hybrids between them. A 1037 bp fragment of the Drasl gene of the D. virilis genome was used as a probe. The gene sequence is localized to the region of the disk 25 A-B on the chromosome 2 of the polytene chromosome map of D. virilis.

Alternative DNA Structures In Vivo: Molecular Evidence and Remaining Questions.

Duplex DNA naturally folds into a right-handed double helix in physiological conditions. Some sequences of unusual base composition may nevertheless form alternative structures, as was shown for many repeated sequences in vitro However, evidence for the formation of noncanonical structures in living cells is difficult to gather. It mainly relies on genetic assays demonstrating their function in vivo or through genetic instability reflecting particular properties of such structures. Efforts were made to reveal their existence directly in a living cell, mainly by generating antibodies specific to secondary structures or using chemical ligands selected for their affinity to these structures. Among secondary structure-forming DNAs are G-quadruplexes, human fragile sites containing minisatellites, AT-rich regions, inverted repeats able to form cruciform structures, hairpin-forming CAG/CTG triplet repeats, and triple helices formed by homopurine-homopyrimidine GAA/TTC trinucleotide repeats. Many of these alternative structures are involved in human pathologies, such as neurological or developmental disorders, as in the case of trinucleotide repeats, or cancers triggered by translocations linked to fragile sites. This review will discuss and highlight evidence supporting the formation of alternative DNA structures in vivo and will emphasize the role of the mismatch repair machinery in binding mispaired DNA duplexes, triggering genetic instability.

PASA: Identifying More Credible Structural Variants of Hedou12.

Although plenty of structural variant detecting approaches for human genomes can be looked up in the literatures, little has been acknowledged on the effectiveness of those structural variant softwares for plant genomes. Moreover, it has been demonstrated frequent occurrences for those structural variant detecting softwares to find too many false structural variants. In this paper, we devote to detect deletions, insertions, and inversions, in total of three kinds of structural variants occurring in Hedou12 genome in contrast to Williams82 genome. To find more potential structural variants, we try to develop new principles to detect discordant and split read map sets supporting structural variants. Aiming to enhance the precision of structural variant detections, we propose two new sequencing characteristic based probability models, which use the sequencing parameters of Hedou12 genome as well as the parameters for Hedou12 paired-end reads to be aligned onto Williams82, to evaluate the probability for a potential structural variant to occur in. To remove the false members from those potential structural variants, we propose a set cover problem model to describe formally on which potential structural variants it should accept to achieve as high as possible a probability summation. This will achieve a solution with more credible structural variants, which can be verified by comparing with DELLY version 0.5.8 and LUMPY version 0.2.2.3. Our algorithm has been verified to be able to find deletions, insertions, and inversions in Hedou12 in contrast to Williams82 DELLY as well as LUMPY fails to find.

Large-scale chromosome flip-flop reversible inversion mediates phenotypic switching of expression of antibiotic resistance in lactococci.

Bacteria can gain resistance to antimicrobials by acquiring and expressing genetic elements that encode resistance determinants such as efflux pumps and drug-modifying enzymes, thus hampering treatment of infection. Previously we showed that acquisition of spectinomycin resistance in a lactococcal strain was correlated with a reversible genomic inversion, but the precise location and the genes affected were unknown. Here we use long-read whole-genome sequencing to precisely define the genomic inversion and we use quantitative PCR to identify associated changes in gene expression levels. The boundaries of the inversion fall within two identical copies of a prophage-like sequence, located on the left and right replichores; this suggests possible mechanisms for inversion through homologous recombination or prophage activity. The inversion is asymmetrical in respect of the axis between the origin and terminus of the replication and modulates the expression of a SAM-dependent methyltransferase, whose heterologous expression confers resistance to spectinomycin in lactococci and that is up-regulated on exposure to spectinomycin. This study provides one of the first examples of phase variation via large-scale chromosomal inversions that confers a switch in antimicrobial resistance in bacteria and the first outside of Staphylococcus aureus.

Acquisition of inverted GSTM exons by an intron of primate GSTM5 gene.

The human GSTM gene family is composed of five gene members, GSTM1-5, and plays an important role in detoxification. In this study, the human GSTM5 gene was found to have a long inverted repeat (LIR) in intron 5. The LIR is able to form a stem-loop structure with a 31-bp stem and a 9-nt loop. The intronic LIR was also identified in other primates but not in non-primates. The human and chimpanzee LIRs had undergone compensating mutations that make the stem loop more stable, suggesting a functional role for the LIR. Sequence homology showed that the LIR was actually a part of inverted exons acquired by the intron. Results of phylogenetic analysis indicate that the inverted exons were derived from exon 5 of GSTM4 and exon 5 of GSTM1. The intronic LIR and inverted GSTM exons can probably introduce complexity in the expression of GSTM gene family.

DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer.

DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3' of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified.