Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR.

Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.1 to 742 kb flanked by inverted repeats (IRs) up to 134 kb long. Of those, we validated 13 inversions predicted by different genome-wide techniques. In addition, we obtained new experimental human population information across 95 African, European, and East Asian individuals for 16 inversions, including four already validated variants without high-throughput genotyping methods. Through comparison with previous data, independent replicates and both inversion breakpoints, we demonstrate that the technique is highly accurate and reproducible. Most studied inversions are widespread across continents, and their frequency is negatively correlated with genetic length. Moreover, all except two show clear signs of being recurrent, and we could better define the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, the generated genotypes have allowed us to check inversion functional effects, validating gene expression differences reported before for two inversions and finding new candidate associations. Therefore, the developed methodology makes it possible to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

Evolutionary and functional impact of common polymorphic inversions in the human genome.

Inversions are one type of structural variants linked to phenotypic differences and adaptation in multiple organisms. However, there is still very little information about polymorphic inversions in the human genome due to the difficulty of their detection. Here, we develop a new high-throughput genotyping method based on probe hybridization and amplification, and we perform a complete study of 45 common human inversions of 0.1-415 kb. Most inversions promoted by homologous recombination occur recurrently in humans and great apes and they are not tagged by SNPs. Furthermore, there is an enrichment of inversions showing signatures of positive or balancing selection, diverse functional effects, such as gene disruption and gene-expression changes, or association with phenotypic traits. Therefore, our results indicate that the genome is more dynamic than previously thought and that human inversions have important functional and evolutionary consequences, making possible to determine for the first time their contribution to complex traits.

Computational Sequence Analysis of Inversion Breakpoint Regions in the Cactophilic Drosophila mojavensis Lineage.

We investigated rates of chromosomal evolution in Drosophila mojavensis using whole-genome sequence information from D. mojavensis, Drosophila buzzatii, and Drosophila virilis. Drosophila mojavensis is a cactophilic species of the repleta group living under extreme ecological conditions in the deserts of the Southwestern United States and Northwestern México. The genome of D. buzzatii, another member of the repleta group, was recently sequenced and the largest scaffolds anchored to all chromosomes using diverse procedures. Chromosome organization between D. mojavensis and D. buzzatii was compared using MUMmer and GRIMM software. Our results corroborate previous cytological analyses that indicated chromosome 2 differed between these 2 species by 10 inversions, chromosomes X and 5 differed by one inversion each, and chromosome 4 was homosequential. In contrast, we found that chromosome 3 differed by 5 inversions instead of the expected 2 that were previously inferred by cytological analyses. Thirteen of these inversions occurred in the D. mojavensis lineage: 12 are fixed and one of them is a polymorphic inversion previously described in populations from Sonora and Baja California, México. We previously investigated the breakpoints of chromosome 2 inversions fixed in D. mojavensis. Here we characterized the breakpoint regions of the 5 inversions found in chromosome 3 in order to infer the molecular mechanism that generated each inversion and its putative functional consequences. Overall, our results reveal a number of gene alterations at the inversion breakpoints with putative adaptive consequences that point to natural selection as the cause for fast chromosomal evolution in D. mojavensis.

Exploration of the Drosophila buzzatii transposable element content suggests underestimation of repeats in Drosophila genomes.

BACKGROUND: Many new Drosophila genomes have been sequenced in recent years using new-generation sequencing platforms and assembly methods. Transposable elements (TEs), being repetitive sequences, are often misassembled, especially in the genomes sequenced with short reads. Consequently, the mobile fraction of many of the new genomes has not been analyzed in detail or compared with that of other genomes sequenced with different methods, which could shed light into the understanding of genome and TE evolution. Here we compare the TE content of three genomes: D. buzzatii st-1, j-19, and D. mojavensis. RESULTS: We have sequenced a new D. buzzatii genome (j-19) that complements the D. buzzatii reference genome (st-1) already published, and compared their TE contents with that of D. mojavensis. We found an underestimation of TE sequences in Drosophila genus NGS-genomes when compared to Sanger-genomes. To be able to compare genomes sequenced with different technologies, we developed a coverage-based method and applied it to the D. buzzatii st-1 and j-19 genome. Between 10.85 and 11.16 % of the D. buzzatii st-1 genome is made up of TEs, between 7 and 7,5 % of D. buzzatii j-19 genome, while TEs represent 15.35 % of the D. mojavensis genome. Helitrons are the most abundant order in the three genomes. CONCLUSIONS: TEs in D. buzzatii are less abundant than in D. mojavensis, as expected according to the genome size and TE content positive correlation. However, TEs alone do not explain the genome size difference. TEs accumulate in the dot chromosomes and proximal regions of D. buzzatii and D. mojavensis chromosomes. We also report a significantly higher TE density in D. buzzatii and D. mojavensis X chromosomes, which is not expected under the current models. Our easy-to-use correction method allowed us to identify recently active families in D. buzzatii st-1 belonging to the LTR-retrotransposon superfamily Gypsy.

Reassignment of Drosophila willistoni Genome Scaffolds to Chromosome II Arms.

Drosophila willistoni is a geographically widespread Neotropical species. The genome of strain Gd-H4-1 from Guadeloupe Island (Caribbean) was sequenced in 2007 as part of the 12 Drosophila Genomes Project. The assembled scaffolds were joined based on conserved linkage and assigned to polytene chromosomes based on a handful of genetic and physical markers. This paucity of markers was particularly striking in the metacentric chromosome II, comprised two similarly sized arms, IIL and IIR, traditionally considered homologous to Muller elements C and B, respectively. In this paper we present the cytological mapping of 22 new gene markers to increase the number of markers mapped by in situ hybridization and to test the assignment of scaffolds to the polytene chromosome II arms. For this purpose, we generated, by polymerase chain reaction amplification, one or two gene probes from each scaffold assigned to the chromosome II arms and mapped these probes to the Gd-H4-1 strain's polytene chromosomes by nonfluorescent in situ hybridization. Our findings show that chromosome arms IIL and IIR correspond to Muller elements B and C, respectively, directly contrasting the current homology assignments in D. willistoni and constituting a major reassignment of the scaffolds to chromosome II arms.

Structural and sequence diversity of the transposon Galileo in the Drosophila willistoni genome.

BACKGROUND: Galileo is one of three members of the P superfamily of DNA transposons. It was originally discovered in Drosophila buzzatii, in which three segregating chromosomal inversions were shown to have been generated by ectopic recombination between Galileo copies. Subsequently, Galileo was identified in six of 12 sequenced Drosophila genomes, indicating its widespread distribution within this genus. Galileo is strikingly abundant in Drosophila willistoni, a neotropical species that is highly polymorphic for chromosomal inversions, suggesting a role for this transposon in the evolution of its genome. RESULTS: We carried out a detailed characterization of all Galileo copies present in the D. willistoni genome. A total of 191 copies, including 133 with two terminal inverted repeats (TIRs), were classified according to structure in six groups. The TIRs exhibited remarkable variation in their length and structure compared to the most complete copy. Three copies showed extended TIRs due to internal tandem repeats, the insertion of other transposable elements (TEs), or the incorporation of non-TIR sequences into the TIRs. Phylogenetic analyses of the transposase (TPase)-encoding and TIR segments yielded two divergent clades, which we termed Galileo subfamilies V and W. Target-site duplications (TSDs) in D. willistoni Galileo copies were 7- or 8-bp in length, with the consensus sequence GTATTAC. Analysis of the region around the TSDs revealed a target site motif (TSM) with a 15-bp palindrome that may give rise to a stem-loop secondary structure. CONCLUSIONS: There is a remarkable abundance and diversity of Galileo copies in the D. willistoni genome, although no functional copies were found. The TIRs in particular have a dynamic structure and extend in different ways, but their ends (required for transposition) are more conserved than the rest of the element. The D. willistoni genome harbors two Galileo subfamilies (V and W) that diverged ~9 million years ago and may have descended from an ancestral element in the genome. Galileo shows a significant insertion preference for a 15-bp palindromic TSM.

Tetris is a foldback transposon that provided the building blocks for an emerging satellite DNA of Drosophila virilis.

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The ß-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs.

Genomics of ecological adaptation in cactophilic Drosophila.

Cactophilic Drosophila species provide a valuable model to study gene-environment interactions and ecological adaptation. Drosophila buzzatii and Drosophila mojavensis are two cactophilic species that belong to the repleta group, but have very different geographical distributions and primary host plants. To investigate the genomic basis of ecological adaptation, we sequenced the genome and developmental transcriptome of D. buzzatii and compared its gene content with that of D. mojavensis and two other noncactophilic Drosophila species in the same subgenus. The newly sequenced D. buzzatii genome (161.5 Mb) comprises 826 scaffolds (>3 kb) and contains 13,657 annotated protein-coding genes. Using RNA sequencing data of five life-stages we found expression of 15,026 genes, 80% protein-coding genes, and 20% noncoding RNA genes. In total, we detected 1,294 genes putatively under positive selection. Interestingly, among genes under positive selection in the D. mojavensis lineage, there is an excess of genes involved in metabolism of heterocyclic compounds that are abundant in Stenocereus cacti and toxic to nonresident Drosophila species. We found 117 orphan genes in the shared D. buzzatii-D. mojavensis lineage. In addition, gene duplication analysis identified lineage-specific expanded families with functional annotations associated with proteolysis, zinc ion binding, chitin binding, sensory perception, ethanol tolerance, immunity, physiology, and reproduction. In summary, we identified genetic signatures of adaptation in the shared D. buzzatii-D. mojavensis lineage, and in the two separate D. buzzatii and D. mojavensis lineages. Many of the novel lineage-specific genomic features are promising candidates for explaining the adaptation of these species to their distinct ecological niches.

A divergent P element and its associated MITE, BuT5, generate chromosomal inversions and are widespread within the Drosophila repleta species group.

The transposon BuT5 caused two chromosomal inversions fixed in two Drosophila species of the repleta group, D. mojavensis and D. uniseta. BuT5 copies are approximately 1-kb long, lack any coding capacity, and do not resemble any other transposable element (TE). Because of its elusive features, BuT5 has remained unclassified to date. To fully characterize BuT5, we carried out bioinformatic similarity searches in available sequenced genomes, including 21 Drosophila species. Significant hits were only recovered for D. mojavensis genome, where 48 copies were retrieved, 22 of them approximately 1-kb long. Polymerase chain reaction (PCR) and dot blot analyses on 54 Drosophila species showed that BuT5 is homogeneous in size and has a widespread distribution within the repleta group. Thus, BuT5 can be considered as a miniature inverted-repeat TE. A detailed analysis of the BuT5 hits in D. mojavensis revealed three partial copies of a transposon with ends very similar to BuT5 and a P-element-like transposase-encoding region in between. A putatively autonomous copy of this P element was isolated by PCR from D. buzzatii. This copy is 3,386-bp long and possesses a seven-exon gene coding for an 822-aa transposase. Exon-intron boundaries were confirmed by reverse transcriptase-PCR experiments. A phylogenetic tree built with insect P superfamily transposases showed that the D. buzzatii P element belongs to an early diverging lineage within the P-element family. This divergent P element is likely the master transposon mobilizing BuT5. The BuT5/P element partnership probably dates back approximately 16 Ma and is the ultimate responsible for the generation of the two chromosomal inversions in the Drosophila repleta species group.

Testing chromosomal phylogenies and inversion breakpoint reuse in Drosophila. The martensis cluster revisited.

The chromosomal relationships of the four martensis cluster species are among the most complex and intricate within the entire Drosophila repleta group, due to the so-called sharing of inversions. Here, we have revised these relationships using comparative mapping of bacterial artificial chromosome (BAC) clones on the salivary gland chromosomes. A physical map of chromosome 2 of Drosophila uniseta (one of the cluster members) was generated by in situ hybridization of 82 BAC clones from the physical map of the Drosophila buzzatii genome (an outgroup that represents the ancestral arrangement). By comparing the marker positions, we determined the number, order, and orientation of conserved chromosomal segments between chromosome 2 of D. buzzatii and D. uniseta. GRIMM software was used to infer that a minimum of five chromosomal inversions are necessary to transform the chromosome 2 of D. buzzatii into that of D. uniseta. Two of these inversions have been overlooked in previous cytological analyses. The five fixed inversions entail two breakpoint reuses because only nine syntenic segments and eight interruptions were observed. We tested for the presence of the five inversions fixed in D. uniseta in the other three species of the martensis cluster by in situ hybridization of eight breakpoint-bearing BAC clones. The results shed light on the chromosomal phylogeny of the martensis cluster, yet leave a number of questions open.

The transposon Galileo generates natural chromosomal inversions in Drosophila by ectopic recombination.

BACKGROUND: Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii. METHODOLOGY/PRINCIPAL FINDINGS: To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z(3). In the non inverted chromosome, the 2z(3) distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric. CONCLUSIONS/SIGNIFICANCE: Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.

A BAC-based physical map of the Drosophila buzzatii genome.

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps, and provide useful resources for sequencing entire genomes. Drosophila buzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation, and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and an approximately 18x expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be approximately 23x. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9555 clones, and assembled them using FingerPrint Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7788 clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.