A strategy for complete telomere-to-telomere assembly of ciliate macronuclear genome using ultra-high coverage Nanopore data.
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ABSTRACT:
SUBMITTER: Wang G
PROVIDER: S-EPMC8060514 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
| S-EPMC1557398 | biostudies-literature
Project description:After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
| S-EPMC7484160 | biostudies-literature
Project description:Synthetic DNA is becoming an attractive substrate for digital data storage due to its density, durability, and relevance in biological research. A major challenge in making DNA data storage a reality is that reading DNA back into data using sequencing by synthesis remains a laborious, slow and expensive process. Here, we demonstrate successful decoding of 1.67 megabytes of information stored in short fragments of synthetic DNA using a portable nanopore sequencing platform. We design and validate an assembly strategy for DNA storage that drastically increases the throughput of nanopore sequencing. Importantly, this assembly strategy is generalizable to any application that requires nanopore sequencing of small DNA amplicons.
| S-EPMC6610119 | biostudies-literature
Project description:BACKGROUND: Nyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement. RESULTS: We examined the structure of these gene-sized macronuclear chromosomes in N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences (4,242) and cDNAs (5,484) and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCC)n, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha) and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21-29 nucleotides), and a significant fraction (1/3) of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway. CONCLUSION: The combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.
| S-EPMC2633312 | biostudies-literature
Project description:We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.
| S-EPMC5889714 | biostudies-literature
Project description:BackgroundThe larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved.ResultsHere, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is contained within five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups in OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome.ConclusionsWe show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica's highly polymorphic genome. This genome assembly opens up the possibility of cross-genome comparison between O. dioica populations, as well as of studies of chromosomal evolution in this lineage.
| S-EPMC8008620 | biostudies-literature
Project description:Ultra-low coverage sequencing (ULCS) is one of the most promising strategies for sequencing based clinical application. These clinical applications, especially prenatal diagnosis, have a strict requirement of turn-around-time; therefore, the application of ULCS is restricted by current high throughput sequencing platforms. Recently, the emergence of rapid sequencing platforms, such as MiSeq and Ion Proton, brings ULCS strategy into a new era. The comparison of their performance could shed lights on their potential application in large-scale clinic trials. In this study, we performed ULCS (<0.1X coverage) on both MiSeq and Ion Proton platforms for 18 spontaneous abortion fetuses carrying aneuploidy and compared their performance on different levels. Overall basic data and GC bias showed no significant difference between these two platforms. We also found the sex and aneuploidy detection indicated 100% sensitivity and 100% specificity on both platforms. Our study generated essential data from these two rapid sequencing platforms, which provides useful reference for later research and potentially accelerates the clinical applications of ULCS.
| S-EPMC3961333 | biostudies-literature
Project description:Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of "green" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name "Candidatus Holospora parva" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.
| S-EPMC5161471 | biostudies-literature
Project description:How to achieve protein diversity by genome and transcriptome processing is essential for organismal complexity and adaptation. The present work identifies that the macronuclear genome of Halteria grandinella, a cosmopolitan unicellular eukaryote, is composed almost entirely of gene-sized nanochromosomes with extremely short nongenic regions. This challenges our usual understanding of chromosomal structure and suggests the possibility of novel mechvanisms in transcriptional regulation. Comprehensive analysis of multiple data sets reveals that Halteria transcription dynamics are influenced by: (i) nonuniform nanochromosome copy numbers correlated with gene-expression level; (ii) dynamic alterations at both the DNA and RNA levels, including alternative internal eliminated sequence (IES) deletions during macronucleus formation and large-scale alternative splicing in transcript maturation; and (iii) extremely short 5' and 3' untranslated regions (UTRs) and universal TATA box-like motifs in the compact 5' subtelomeric regions of most chromosomes. This study broadens the view of ciliate biology and the evolution of unicellular eukaryotes, and identifies Halteria as one of the most compact known eukaryotic genomes, indicating that complex cell structure does not require complex gene architecture.
| S-EPMC7858049 | biostudies-literature
Project description:Polyploidy can provide adaptive advantages and drive evolution. Amitotic division of the polyploid macronucleus (MAC) in ciliates acts as a nonsexual genetic mechanism to enhance adaptation to stress conditions and thus provides a unique model to investigate the evolutionary role of polyploidy. Mutation is the primary source of the variation responsible for evolution and adaptation; however, to date, de novo mutations that occur in ciliate MAC genomes during these processes have not been characterized and their biological impacts are undefined. Here, we carried out long-term evolution experiments to directly explore de novo MAC mutations and their molecular features in the model ciliate, Tetrahymena thermophila. A simple but effective method was established to detect base-substitution mutations in evolving populations whereas filtering out most of the false positive base-substitutions caused by repetitive sequences and the programmed genome rearrangements. The detected mutations were rigorously validated using the MassARRAY system. Validated mutations showed a strong G/C→A/T bias, consistent with observations in other species. Moreover, a progressive increase in growth rate of the evolving populations suggested that some of these mutations might be responsible for cell fitness. The established mutation identification and validation methods will be an invaluable resource to make ciliates an important model system to study the role of polyploidy in evolution.
| S-EPMC7788487 | biostudies-literature