scholarly journals The human ribosomal DNA array is composed of highly homogenized tandem clusters

2021 ◽  
Author(s):  
Yutaro Hori ◽  
Akira Shimamoto ◽  
Takehiko Kobayashi
Keyword(s):  
Ribosomal Dna ◽  
Copy Number ◽  
Large Scale ◽  
Human Cells ◽  
Nanopore Sequencing ◽  
Cellular Functions ◽  
Rdna Cluster ◽  
Repetitive Nature ◽  
Long Read ◽  
Rdna Copy

The structure of the human ribosomal DNA (rDNA) cluster has traditionally been hard to analyze owing to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to study the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its noncoding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all the noncoding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. In addition, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies compared with control normal cells, although the rate was much lower than previously reported using a fiber-FISH method. Collectively, our results clarify the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenizations to ensure sequence quality and maintenance of active copies for cellular functions.

scholarly journals The human ribosomal RNA gene is composed of highly homogenized tandem clusters

2021 ◽  
Author(s):  
Yutaro Hori ◽  
Akira Shimamoto ◽  
Takehiko Kobayashi
Keyword(s):  
Ribosomal Rna ◽  
Copy Number ◽  
Large Scale ◽  
Human Cells ◽  
Gene Clustering ◽  
Nanopore Sequencing ◽  
Coding Region ◽  
Ribosomal Rna Gene ◽  
Coding Regions ◽  
Rdna Copy

The structure of the human ribosomal RNA gene clustering region (rDNA) has traditionally been hard to analyze due to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to approach the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its non-coding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all of the non-coding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. Lastly, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies as compared with control normal cells, although the rate was much lower than previously reported using a Fiber FISH method. Collectively, our results alter the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenization to ensure sequence quality and maintenance of active copies for cellular functions.

scholarly journals Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research

2020 ◽  
Vol 8 (3) ◽  
pp. 316 ◽  
Author(s):  
Yurui Wang ◽  
Yaohan Jiang ◽  
Yongqiang Liu ◽  
Yuan Li ◽  
Laura A. Katz ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
Ribosomal Dna ◽  
Copy Number ◽  
Sequence Variation ◽  
Small Subunit ◽  
Microbial Eukaryotes ◽  
Number Variation ◽  
Small Subunit Ribosomal Dna ◽  
Rdna Copy ◽  
Rdna Copy Number

While nuclear small subunit ribosomal DNA (nSSU rDNA) is the most commonly-used gene marker in studying phylogeny, ecology, abundance, and biodiversity of microbial eukaryotes, mitochondrial small subunit ribosomal DNA (mtSSU rDNA) provides an alternative. Recently, both copy number variation and sequence variation of nSSU rDNA have been demonstrated for diverse organisms, which can contribute to misinterpretation of microbiome data. Given this, we explore patterns for mtSSU rDNA among 13 selected ciliates (representing five classes), a major component of microbial eukaryotes, estimating copy number and sequence variation and comparing to that of nSSU rDNA. Our study reveals: (1) mtSSU rDNA copy number variation is substantially lower than that for nSSU rDNA; (2) mtSSU rDNA copy number ranges from 1.0 × 104 to 8.1 × 105; (3) a most common sequence of mtSSU rDNA is also found in each cell; (4) the sequence variation of mtSSU rDNA are mainly indels in poly A/T regions, and only half of species have sequence variation, which is fewer than that for nSSU rDNA; and (5) the polymorphisms between haplotypes of mtSSU rDNA would not influence the phylogenetic topology. Together, these data provide more insights into mtSSU rDNA as a powerful marker especially for microbial ecology studies.

scholarly journals Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Albina Nowak ◽  
Omer Murik ◽  
Tzvia Mann ◽  
David A. Zeevi ◽  
Gheona Altarescu
Keyword(s):  
Fabry Disease ◽  
Copy Number ◽  
New Technology ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sanger Sequence ◽  
Gla Gene

AbstractMore than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

scholarly journals The retrotransposon R2 maintains Drosophila ribosomal DNA repeats

2021 ◽  
Author(s):  
Jonathan O Nelson ◽  
Alyssa Slicko ◽  
Yukiko M Yamashita
Keyword(s):  
Ribosomal Dna ◽  
Copy Number ◽  
Double Strand Breaks ◽  
Copy Number Loss ◽  
Dna Repeats ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Male Germline ◽  
Rdna Loci ◽  
Rdna Copy

Ribosomal RNAs (rRNAs) account for 80-90% of all transcripts in eukaryotic cells. To meet this demand, the ribosomal DNA (rDNA) gene that codes for rRNA is tandemly repeated hundreds of times, comprising rDNA loci on eukaryotic chromosomes. This repetitiveness imposes a challenge to maintaining sufficient copy number due to spontaneous intra-chromatid recombination between repetitive units causing copy number loss. The progressive shrinking of rDNA loci from generation to generation could lead to extinction of the lineage, yet the mechanism(s) to counteract spontaneous copy number loss remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for rDNA copy number (CN) maintenance in the Drosophila male germline, despite the perceived disruptive nature of transposable elements. Depletion of R2 led to defective rDNA CN maintenance in multiple contexts, causing a decline in fecundity over generations and eventual extinction of the lineage. Our data suggests that DNA double strand breaks generated by R2 is the initiating event of rDNA CN expansion, stimulating the repair processes proposed to underlie rDNA CN expansion. This study reveals that retrotransposons can provide a benefit to their hosts, contrary to their reputation as genomic parasitic, which may contribute to their widespread success throughout taxa.

scholarly journals Detection of Single Nucleotide and Copy Number Variants in the Fabry Disease-associated GLA Gene Using Nanopore Sequencing

2021 ◽  
Author(s):  
Albina Nowak ◽  
Omer Murik ◽  
Tzvia Mann ◽  
David A. Zeevi ◽  
Gheona Altarescu
Keyword(s):  
Fabry Disease ◽  
Copy Number ◽  
New Technology ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sanger Sequence ◽  
Gla Gene

Abstract Introduction: More than one thousand variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence.Aims: We aimed to design and validate a method for sequencing the GLA gene using long read Oxford Nanopore sequencing technology.Methods: Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long read sequencing of a 13kb PCR amplicon. We used minimap2 to align the long read data and Nanopolish and Sniffles to call variants.Results: All the variants detected by Sanger (including a deep intronic variant) were also detected by long read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology.Conclusions: Our long read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

scholarly journals A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

2014 ◽  
Vol 35 (3) ◽  
pp. 544-554 ◽  
Author(s):  
Inswasti Cahyani ◽  
Andrew G. Cridge ◽  
David R. Engelke ◽  
Austen R. D. Ganley ◽  
Justin M. O'Sullivan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Intergenic Region ◽  
Copy Number ◽  
Rna Polymerase I ◽  
Intergenic Sequence ◽  
Polymerase I ◽  
Rdna Copy ◽  
Rdna Copy Number

The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within theSaccharomyces cerevisiaegenome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e.,RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within theRPA135-tK(CUU)Pintergenic region that is involved in the interaction. We demonstrate that theRPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not governRPA135transcription. Instead, replacement of a 605-bp region within theRPA135-tK(CUU)Pintergenic region results in a reduction in theRPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between theRPA135-tK(CUU)Pand rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome.

scholarly journals Dosage-sensitivity of human transcription factor genes

2019 ◽  
Author(s):  
Zhihua Ni ◽  
Xiao-Yu Zhou ◽  
Sidra Aslam ◽  
Deng-Ke Niu
Keyword(s):  
Transcription Factor ◽  
Copy Number ◽  
Large Scale ◽  
Gene Dosage ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Protein Coding ◽  
Genomic Study ◽  
Genomic Studies ◽  
Dosage Sensitivity

AbstractChanges in the copy number of protein-coding genes would lead to detrimental effects if the consequent changes in protein concentration disrupt essential cellular functions. Large-scale genomic studies have identified thousands of dosage-sensitive genes in human genome. We are interested in the dosage-sensitivity of transcription factor (TF) genes whose products are essential for the growth, division and differentiation of cells by regulating the expression of the genetic information encoded in the genome. We first surveyed the enrichment of human TF genes in four recently curated datasets of dosage-sensitive genes, including the haploinsufficient genes identified by a large-scale genomic study, the haploinsufficient genes predicted by a machine learning approach, the genes with conserved copy number across mammals, and the ohnologs. Then we selected the dosage-sensitive genes that are present in all the four dataset and regarded them as the most reliable dosage-sensitive genes, and the genes that are absent from any one of the four datasets as the most reliable dosage-insensitive genes, and surveyed the enrichments of TFs genes in these two datasets. A large number of TF genes were found to be dosage-insensitive, which is beyond the expectation based on the role of TFs. In spite of this, the likeness of TF genes to be dosage-sensitive were supported by five datasets, with the conserved-copy-number genes as the exception. The nuclear receptors are the only one family of TFs whose dosage-sensitivity was consistently supported by all the six datasets. In addition, we found that TF families with very few members are also more likely to be dosage-sensitive while the largest TF family, C2H2-ZF, are most likely dosage-insensitive. The most extensively studied TFs, p53, are not special in dosage-sensitivity. They are significantly enriched in only three datasets. We also confirmed that dosage-sensitive genes generally have long coding sequences, high expression levels and experienced stronger selective pressure. Our results indicate some TFs function in a dose-dependent manner while some other not. Gene dosage changes in some TF families like nuclear receptor would result in disease phenotypes while the effects of such changes in some TFs like C2H2-ZF would be mild.

scholarly journals Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

Science ◽  
2019 ◽  
Vol 366 (6463) ◽  
pp. eaax2083 ◽  
Author(s):  
PingHsun Hsieh ◽  
Mitchell R. Vollger ◽  
Vy Dang ◽  
David Porubsky ◽  
Carl Baker ◽  
...  
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Sequence Data ◽  
Local Population ◽  
Copy Number Variants ◽  
Human Populations ◽  
Single Nucleotide Variants ◽  
Human Genes ◽  
Long Read ◽  
Archaic Introgression

Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.

scholarly journals Copy numbers of 45S and 5S ribosomal DNA arrays lack meaningful correlation in humans

2020 ◽  
Author(s):  
Ashley N. Hall ◽  
Tychele N. Turner ◽  
Christine Queitsch
Keyword(s):  
Copy Number Variation ◽  
Ribosomal Dna ◽  
Copy Number ◽  
1000 Genomes Project ◽  
High Coverage ◽  
1000 Genomes ◽  
Copy Numbers ◽  
Number Variation ◽  
Rdna Copy ◽  
Rdna Copy Number

AbstractThe ribosomal DNA genes are tandemly arrayed in most eukaryotes and exhibit vast copy number variation. There is growing interest in integrating this variation into genotype-phenotype associations. Here, we explored a possible association of rDNA copy number variation with autism spectrum disorder and found no difference between probands and unaffected siblings. However, rDNA copy number estimates from whole genome sequencing are error-prone, so we sought to use pulsed-field gel electrophoresis, a classic gold-standard method, to validate rDNA copy number genotypes. The electrophoresis approach is not readily applicable to the human 45S arrays due to their size and location on five separate chromosomes; however, it should accurately resolve copy numbers for the shorter 5S arrays that reside on a single chromosome. Previous studies reported tightly correlated, concerted copy number variation between the 45S and 5S arrays, which should enable the validation of 45S copy number estimates with CHEF-gel-verified 5S copy numbers. Here, we show that the previously reported strong concerted copy number variation is likely an artifact of variable data quality in the earlier published 1000 Genomes Project sequences. We failed to detect a meaningful correlation between 45S and 5S copy numbers in the large, high-coverage Simons Simplex Collection dataset as well as in the recent high-coverage 1000 Genomes Project sequences. Our findings illustrate the challenge of genotyping repetitive DNA regions accurately and call into question the accuracy of recently published studies of rDNA copy number variation in cancers and aging that relied on diverse publicly available resources for sequence data.

scholarly journals circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Zelin Liu ◽  
Changyu Tao ◽  
Shiwei Li ◽  
Minghao Du ◽  
Yongtai Bai ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Full Length ◽  
Circular Rnas ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Functional Studies ◽  
Sequencing Method ◽  
Long Read ◽  
Fine Tune

Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

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