Mosaic deletions in X-linked dystonia-parkinsonism influence repeat stability and disease onset

Background: While multiple genetic causes of movement disorders have been identified in the past decade, modifying factors of disease expression are still largely unknown for most conditions. X-linked dystonia-parkinsonism (XDP) is an inherited neurodegenerative disease caused by a SINE-VNTR-Alu (SVA)-type retrotransposon insertion that contains a hexanucleotide repeat within an intron of the TAF1 gene. To date, four putative genetic modifiers explain about 65% of variance in age at onset in XDP. However, additional genetic modifiers are conceivably at play in XDP and may include mismatches of the SVA hexanucleotide repeat motif. We aim to identify additional genetic modifiers of XDP expressivity and age at onset (AAO). Methods: Third-generation sequencing of PCR amplicons from XDP patients (n=202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment were used to confirm the presence of identified repeat mismatches. Results: An increased frequency of deletions at the beginning of the hexanucleotide repeat (CCCTCT)n domain was found. Specifically, three deletions at positions 11, 14, and 17 of the TAF1 SVA repeat motif of somatic mosaic origins were detected in different combinations. The most common one was three deletions (1-2-3) at a median frequency 0.425 (IQR:0.42-0.43) and deletions within positions 11 and 14 (1-2-wt) at a median frequency 0.128 (IQR:0.12-0.13). The frequency of deletions at positions 11 and 14 correlated with repeat number (r=-0.48, p=9.5x10-13) and AAO (r=0.34, p=9.5x10-7). The association with AAO still stands when including other modifier genotypes (MSH3 and PMS2) in a regression model. However, the association dissipates when including repeat numbers. Conclusion: We present a novel mosaic repeat motif deletion within the hexanucleotide repeat (CCCTCT)n domain of TAF1 SVA. Our study illustrates: 1) the importance of somatic mosaic genotypes; 2) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; 3) that these variations may remain undetected without assessment of single molecules.

Perspectives in Earthworm Molecular Phylogeny: Recent Advances in Lumbricoidea and Standing Questions

Earthworm systematics have been limited by the small number of taxonomically informative morphological characters and high levels of homoplasy in this group. However, molecular phylogenetic techniques have yielded significant improvements in earthworm taxonomy in the last 15 years. Several different approaches based on the use of different molecular markers, sequencing techniques, and compromises between specimen/taxon coverage and phylogenetic information have recently emerged (DNA barcoding, multigene phylogenetics, mitochondrial genome analysis, transcriptome analysis, targeted enrichment methods, and reduced representation techniques), providing solutions to different evolutionary questions regarding European earthworms. Molecular phylogenetics have led to significant advances being made in Lumbricidae systematics, such as the redefinition or discovery of new genera (Galiciandrilus, Compostelandrilus, Vindoboscolex, Castellodrilus), delimitation and revision of previously existing genera (Kritodrilus, Eophila, Zophoscolex, Bimastos), and changes to the status of subspecific taxa (such as the Allolobophorachaetophora complex). These approaches have enabled the identification of problems that can be resolved by molecular phylogenetics, including the revision of Aporrectodea, Allolobophora, Helodrilus, and Dendrobaena, as well as the examination of small taxa such as Perelia, Eumenescolex, and Iberoscolex. Similar advances have been made with the family Hormogastridae, in which integrative systematics have contributed to the description of several new species, including the delimitation of (formerly) cryptic species. At the family level, integrative systematics have provided a new genus system that better reflects the diversity and biogeography of these earthworms, and phylogenetic comparative methods provide insight into earthworm macroevolution. Despite these achievements, further research should be performed on the Tyrrhenian cryptic complexes, which are of special eco-evolutionary interest. These examples highlight the potential value of applying molecular phylogenetic techniques to other earthworm families, which are very diverse and occupy different terrestrial habitats across the world. The systematic implementation of such approaches should be encouraged among the different expert groups worldwide, with emphasis on collaboration and cooperation.

Application of target enrichment sequencing for population genetic analyses of the obligate plant pathogens Pseudoperonospora cubensis and P. humuli in Michigan

Technological advances in genome sequencing have improved our ability to catalog genomic variation and led to an expansion of the scope and scale of genetic studies. Yet, for agronomically important plant pathogens such as the downy-mildews the scale of genetic studies remains limited. This is, in part, due to the difficulties associated with maintaining obligate pathogens, and the logistical constraints involved in the genotyping of these species. To study the genetic variation of two Pseudoperonospora species (P. cubensis and P. humuli), we describe a targeted enrichment (TE) protocol able to genotype isolates using less than 50 ng of mixed pathogen and plant DNA for library preparation. We enriched 830 target genes across 128 samples and identified 2,514 high-quality SNP variants. We detected significant genetic differentiation (p=0.01) between P. cubensis subpopulations from Cucurbita moschata (clade I) and Cucumis sativus (clade II) in Michigan. No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation. A significant effect of location on the genetic variation of the P. humuli subpopulation was detected in the state (p=0.01). Mantel tests found evidence that the genetic distance among P. humuli samples was associated with the physical distance of the hop yards from which the samples were collected (p=0.005). The differences in the distribution of genetic variation of the P. humuli and P. cubensis subpopulations of Michigan suggest differences in the dispersal of these two species. Our TE protocol provides an additional tool for genotyping obligate pathogens and the execution of new genetic studies

Cas9 targeted enrichment of mobile elements using nanopore sequencing

Mobile element insertions (MEIs) are repetitive genomic sequences that contribute to genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9-targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on-targeted signals and exhibiting a 13.4-54x enrichment over whole-genome approaches. We show an individual flow cell can recover most MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify seventeen non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

Targeted Enrichment of rRNA Gene Tandem Arrays for Ultra-Long Sequencing by Selective Restriction Endonuclease Digestion

Large regions of nearly identical repeats, such as the 45S ribosomal RNA (rRNA) genes of Nucleolus Organizer Regions (NORs), can account for major gaps in sequenced genomes. To assemble these regions, ultra-long sequencing reads that span multiple repeats have the potential to reveal sets of repeats that collectively have sufficient sequence variation to unambiguously define that interval and recognize overlapping reads. Because individual repetitive loci typically represent a small proportion of the genome, methods to enrich for the regions of interest are desirable. Here we describe a simple method that achieves greater than tenfold enrichment of Arabidopsis thaliana 45S rRNA gene sequences among ultra-long Oxford Nanopore Technology sequencing reads. This method employs agarose-embedded genomic DNA that is subjected to restriction endonucleases digestion using a cocktail of enzymes predicted to be non-cutters of rRNA genes. Most of the genome is digested into small fragments that diffuse out of the agar plugs, whereas rRNA gene arrays are retained. In principle, the approach can also be adapted for sequencing other repetitive loci for which gaps exist in a reference genome.

Cas9 targeted enrichment of mobile elements using nanopore sequencing

Mobile element insertions (MEIs) are highly repetitive genomic sequences that contribute to inter- and intra-individual genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9 targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on targeted signals. We show an individual flow cell can recover a remarkable fraction of MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify twenty-one non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

Targeted enrichment of novel chloroplast-based probes reveals a large-scale phylogeny of 412 bamboos

Background The subfamily Bambusoideae belongs to the grass family Poaceae and has significant roles in culture, economy, and ecology. However, the phylogenetic relationships based on large-scale chloroplast genomes (CpGenomes) were elusive. Moreover, most of the chloroplast DNA sequencing methods cannot meet the requirements of large-scale CpGenome sequencing, which greatly limits and impedes the in-depth research of plant genetics and evolution. Results To develop a set of bamboo probes, we used 99 high-quality CpGenomes with 6 bamboo CpGenomes as representative species for the probe design, and assembled 15 M unique sequences as the final pan-chloroplast genome. A total of 180,519 probes for chloroplast DNA fragments were designed and synthesized by a novel hybridization-based targeted enrichment approach. Another 468 CpGenomes were selected as test data to verify the quality of the newly synthesized probes and the efficiency of the probes for chloroplast capture. We then successfully applied the probes to synthesize, enrich, and assemble 358 non-redundant CpGenomes of woody bamboo in China. Evaluation analysis showed the probes may be applicable to chloroplasts in Magnoliales, Pinales, Poales et al. Moreover, we reconstructed a phylogenetic tree of 412 bamboos (358 in-house and 54 published), supporting a non-monophyletic lineage of the genus Phyllostachys. Additionally, we shared our data by uploading a dataset of bamboo CpGenome into CNGB (https://db.cngb.org/search/project/CNP0000502/) to enrich resources and promote the development of bamboo phylogenetics. Conclusions The development of the CpGenome enrichment pipeline and its performance on bamboos recommended an inexpensive, high-throughput, time-saving and efficient CpGenome sequencing strategy, which can be applied to facilitate the phylogenetics analysis of most green plants.