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

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.

Download Full-text

A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations

10.1101/2021.01.21.427686 ◽

2021 ◽

Author(s):

Diksha Sharma ◽

Sylvie Hermann-Le Denmat ◽

Nicholas J. Matzke ◽

Katherine Hannan ◽

Ross D. Hannan ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Copy Number Variation ◽

Ribosomal Dna ◽

Copy Number ◽

Whole Genome Sequence ◽

Whole Genome ◽

Copy Numbers ◽

Number Variation ◽

Rdna Copy ◽

Rdna Copy Number

AbstractRibosomal DNA genes (rDNA) encode the major ribosomal RNAs (rRNA) and in eukaryotic genomes are typically present as one or more arrays of tandem repeats. Species have characteristic rDNA copy numbers, ranging from tens to thousands of copies, with the number thought to be redundant for rRNA production. However, the tandem rDNA repeats are prone to recombination-mediated changes in copy number, resulting in substantial intra-species copy number variation. There is growing evidence that these copy number differences can have phenotypic consequences. However, we lack a comprehensive understanding of what determines rDNA copy number, how it evolves, and what the consequences are, in part because of difficulties in quantifying copy number. Here, we developed a genomic sequence read approach that estimates rDNA copy number from the modal coverage of the rDNA and whole genome to help overcome limitations in quantifying copy number with existing mean coverage-based approaches. We validated our method using strains of the yeast Saccharomyces cerevisiae with previously-determined rDNA copy numbers, and then applied our pipeline to investigate rDNA copy number in a global sample of 788 yeast isolates. We found that wild yeast have a mean copy number of 92, consistent with what is reported for other fungi but much lower than in laboratory strains. We also show that different populations have different rDNA copy numbers. These differences can partially be explained by phylogeny, but other factors such as environment are also likely to contribute to population differences in copy number. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.Author summaryThe ribosomal RNA gene repeats (rDNA) form large tandem repeat arrays in most eukaryote genomes. Their tandem arrangement makes the rDNA prone to copy number variation, and there is increasing evidence that this copy number variation has phenotypic consequences. However, difficulties in measuring rDNA copy number hamper investigation into rDNA copy number dynamics and their significance. Here we developed a novel bioinformatics method for measuring rDNA copy number from whole genome sequence data that is based on the modal sequence read coverage. We established parameters for optimal performance of the method and validated it using yeast strains of known rDNA copy numbers. We then applied the method to a dataset of almost 800 global yeast isolates and demonstrate that yeast populations have different rDNA copy numbers that partially correlate with phylogeny. Our work provides a simple and accurate method for determining rDNA copy number that leverages the growing number of whole genome datasets, and highlights the dynamic nature of rDNA copy number.

Download Full-text

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

Microorganisms ◽

10.3390/microorganisms8030316 ◽

2020 ◽

Vol 8 (3) ◽

pp. 316 ◽

Cited By ~ 3

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.

Download Full-text

Genetic context and proliferation determine contrasting patterns of copy number amplification and loss for 5S and 45S ribosomal DNA arrays in cancer

10.1101/154120 ◽

2017 ◽

Cited By ~ 1

Author(s):

Meng Wang ◽

Bernardo Lemos

Keyword(s):

Copy Number Variation ◽

Ribosomal Dna ◽

Copy Number ◽

5S Rdna ◽

45S Rdna ◽

Rdna Array ◽

Number Variation ◽

Proliferating Cell ◽

Rdna Copy ◽

Rdna Copy Number

AbstractThe multicopy 45S ribosomal DNA (45S rDNA) array gives origin to the nucleolus, the first discovered nuclear organelle, site of Poll I 45S rRNA transcription and key regulator of cellular metabolism, DNA repair response, genome stability, and global epigenetic states. The multicopy 5S ribosomal DNA array (5S rDNA) is located on a separate chromosome, encodes the 5S rRNA transcribed by Pol III, and exhibits concerted copy number variation (cCNV) with the 45S rDNA array in human blood. Here we combined genomic data from >700 tumors and normal tissues to provide a portrait of ribosomal DNA variation in human tissues and cancers of diverse mutational signatures. We show that most cancers undergo coupled 5S rDNA array amplification and 45S rDNA loss, with abundant inter-individual variation in rDNA copy number of both arrays, and concerted modulation of 5S-45S copy number in some but not all tissues. Analysis of genetic context revealed associations between the presence of specific somatic alterations, such as P53 mutations in stomach and lung adenocarcinomas, and coupled 5S gain / 45S loss. Finally, we show that increased proliferation rates along cancer lineages can partially explain contrasting copy number changes in the 5S and 45S rDNA arrays. We suggest that 5S rDNA amplification facilitates increased ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in highly proliferating tumor cells. Our results highlight the tissue- specificity of concerted copy number variation and uncover contrasting changes in 5S and 45S rDNA copy number along rapidly proliferating cell lineages.Lay SummaryThe 45S and 5S ribosomal DNA (rDNA) arrays contain hundreds of rDNA copies, with substantial variability across individuals and species. Although physically unlinked, both arrays exhibit concerted copy number variation. However, whether concerted copy number is universally observed across all tissues is unknown. It also remains unknown if rDNA copy number may vary in tissues and cancer lineages. Here we showed that most cancers undergo coupled 5S rDNA array amplification and 45S rDNA loss, and concerted 5S-45S copy number variation in some but not all tissues. The coupled 5S amplification and 45S loss is associated with the presence of certain somatic genetic variations, as well as increased cancerous cell proliferation rate. Our research highlights the tissue- specificity of concerted copy number variation and uncover contrasting changes in rDNA copy number along rapidly proliferating cell lineages. Our observations raise the prospects of using 5S and 45S ribosomal DNA states as indicators of cancer status and targets in new strategies for cancer therapy.

Download Full-text

Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2017.0425 ◽

2017 ◽

Vol 284 (1859) ◽

pp. 20170425 ◽

Cited By ~ 42

Author(s):

Chundi Wang ◽

Tengteng Zhang ◽

Yurui Wang ◽

Laura A. Katz ◽

Feng Gao ◽

...

Keyword(s):

Single Cell ◽

Copy Number ◽

Sequence Variation ◽

Ssu Rdna ◽

Copy Numbers ◽

Number Variation ◽

The Impact ◽

Rdna Copy ◽

Rdna Polymorphism ◽

Rdna Copy Number

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella , Blepharisma americanum and Strombidium stylifer . We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

Download Full-text

Spontaneous rDNA copy number variation modulates Sir2 levels and epigenetic gene silencing

Genes & Development ◽

10.1101/gad.340205 ◽

2005 ◽

Vol 19 (10) ◽

pp. 1199-1210 ◽

Cited By ~ 50

Author(s):

A. H. Michel

Keyword(s):

Copy Number Variation ◽

Gene Silencing ◽

Copy Number ◽

Epigenetic Gene Silencing ◽

Number Variation ◽

Rdna Copy ◽

Rdna Copy Number

Download Full-text

Genome‐based estimates of fungal rDNA copy number variation across phylogenetic scales and ecological lifestyles

Molecular Ecology ◽

10.1111/mec.14995 ◽

2019 ◽

Vol 28 (4) ◽

pp. 721-730 ◽

Cited By ~ 39

Author(s):

Lotus A. Lofgren ◽

Jessie K. Uehling ◽

Sara Branco ◽

Thomas D. Bruns ◽

Francis Martin ◽

...

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Number Variation ◽

Rdna Copy ◽

Rdna Copy Number

Download Full-text

Copy Number Variation detection from 1000 Genomes project exon capture sequencing data

BMC Bioinformatics ◽

10.1186/1471-2105-13-305 ◽

2012 ◽

Vol 13 (1) ◽

pp. 305 ◽

Cited By ~ 21

Author(s):

Jiantao Wu ◽

Krzysztof R Grzeda ◽

Chip Stewart ◽

Fabian Grubert ◽

Alexander E Urban ◽

...

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Sequencing Data ◽

1000 Genomes Project ◽

1000 Genomes ◽

Exon Capture ◽

Number Variation ◽

Copy Number Variation Detection ◽

Capture Sequencing

Download Full-text

Salivary AMY1 Copy Number Variation Modifies Age-Related Type 2 Diabetes Risk

Clinical Chemistry ◽

10.1093/clinchem/hvaa072 ◽

2020 ◽

Vol 66 (5) ◽

pp. 718-726

Author(s):

Yuwei Liu ◽

Caren E Smith ◽

Laurence D Parnell ◽

Yu-Chi Lee ◽

Ping An ◽

...

Keyword(s):

Type 2 Diabetes ◽

Copy Number Variation ◽

Metabolic Pathway ◽

Copy Number ◽

Lipid Lowering ◽

Pathway Enrichment Analysis ◽

Copy Numbers ◽

Number Variation ◽

The Relationship

Abstract Background Copy number variation (CNV) in the salivary amylase gene (AMY1) modulates salivary α-amylase levels and is associated with postprandial glycemic traits. Whether AMY1-CNV plays a role in age-mediated change in insulin resistance (IR) is uncertain. Methods We measured AMY1-CNV using duplex quantitative real-time polymerase chain reaction in two studies, the Boston Puerto Rican Health Study (BPRHS, n = 749) and the Genetics of Lipid-Lowering Drug and Diet Network study (GOLDN, n = 980), and plasma metabolomic profiles in the BPRHS. We examined the interaction between AMY1-CNV and age by assessing the relationship between age with glycemic traits and type 2 diabetes (T2D) according to high or low copy numbers of the AMY1 gene. Furthermore, we investigated associations between metabolites and interacting effects of AMY1-CNV and age on T2D risk. Results We found positive associations of IR with age among subjects with low AMY1-copy-numbers in both studies. T2D was marginally correlated with age in participants with low AMY1-copy-numbers but not with high AMY1-copy-numbers in the BPRHS. Metabolic pathway enrichment analysis identified the pentose metabolic pathway based on metabolites that were associated with both IR and the interactions between AMY1-CNV and age. Moreover, in older participants, high AMY1-copy-numbers tended to be associated with lower levels of ribonic acid, erythronic acid, and arabinonic acid, all of which were positively associated with IR. Conclusions We found evidence supporting a role of AMY1-CNV in modifying the relationship between age and IR. Individuals with low AMY1-copy-numbers tend to have increased IR with advancing age.

Download Full-text