High variability in SSU rDNA gene copy number among planktonic foraminifera revealed by single-cell qPCR

AbstractMetabarcoding has become the workhorse of community ecology. Sequencing a taxonomically informative DNA fragment from environmental samples gives fast access to community composition across taxonomic groups, but it relies on the assumption that the number of sequences for each taxon correlates with its abundance in the sampled community. However, gene copy number varies among and within taxa, and the extent of this variability must therefore be considered when interpreting community composition data derived from environmental sequencing. Here we measured with single-cell qPCR the SSU rDNA gene copy number of 139 specimens of five species of planktonic foraminifera. We found that the average gene copy number varied between of ~4000 to ~50,000 gene copies between species, and individuals of the same species can carry between ~300 to more than 350,000 gene copies. This variability cannot be explained by differences in cell size and considering all plausible sources of bias, we conclude that this variability likely reflects dynamic genomic processes acting during the life cycle. We used the observed variability to model its impact on metabarcoding and found that the application of a correcting factor at species level may correct the derived relative abundances, provided sufficiently large populations have been sampled.

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Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

PLoS ONE ◽

10.1371/journal.pone.0162544 ◽

2016 ◽

Vol 11 (9) ◽

pp. e0162544 ◽

Cited By ~ 16

Author(s):

Marcela Rosato ◽

Aleš Kovařík ◽

Ricardo Garilleti ◽

Josep A. Rosselló

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Land Plants ◽

Gene Copy ◽

45S Rdna ◽

Rdna Gene ◽

Rdna Sites ◽

Early Land

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Nosocomial Outbreak of Carbapenemase-Producing Proteus mirabilis With Two Novel Salmonella Genomic Island 1 Variants Carrying Different blaNDM–1 Gene Copies in China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.800938 ◽

2022 ◽

Vol 12 ◽

Author(s):

Lang Yang ◽

Hong He ◽

Qichao Chen ◽

Kaiying Wang ◽

Yanfeng Lin ◽

...

Keyword(s):

Proteus Mirabilis ◽

Copy Number ◽

Genomic Island ◽

Gene Copy Number ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Gene Copy ◽

Nosocomial Outbreak ◽

Gene Copy Number Variation ◽

Gene Copies

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a blaNDM–1 gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two blaNDM–1 gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-blaNDM–1-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without blaNDM–1 copy number changes. This study characterizes two novel blaNDM–1-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

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Effect of EPSPS Gene Copy Number and Glyphosate Selection on Fitness of Glyphosate-Resistant Bassia Scoparia in The Field

10.21203/rs.3.rs-155310/v1 ◽

2021 ◽

Author(s):

Charlemagne Ajoc Lim ◽

Prashant Jha ◽

Vipan Kumar ◽

Alan T. Dyer

Keyword(s):

Sugar Beet ◽

Seed Production ◽

Great Plains ◽

Copy Number ◽

Gene Copy Number ◽

Reproductive Traits ◽

Gene Copy ◽

Epsps Gene ◽

Gene Copies

Abstract The widespread evolution of glyphosate-resistant (GR) Bassia scoparia in the U.S. Great Plains poses a serious threat to the long-term sustainability of GR sugar beet. Glyphosate resistance in B. scoparia is due to an increase in the EPSPS (5-enolpyruvyl-shikimate-3-phosphate) gene copy number. The variation in EPSPS gene copies among individuals from within a single GR B. scoparia population indicated a differential response to glyphosate selection. We tested the hypothesis of reduced GR B. scoparia fitness (reproductive traits) to increasing glyphosate rates (applied as single or sequential applications) potentially experienced within a GR sugar beet field. The variation in EPSPS gene copy number and total glyphosate rate (single or sequential applications) did not influence any of the reproductive traits of GR B. scoparia, except seed production. Sequential applications of glyphosate with a total rate of 2,214 g ae ha− 1 or higher prevented seed production in B. scoparia plants with 2–4 (low levels of resistance) and 5–6 (moderate levels of resistance) EPSPS gene copies. Timely sequential applications of glyphosate (full recommended rates) can potentially slow down the evolution of GR B. scoparia with low to moderate levels of resistance (2–6 EPSPS gene copies), but any survivors (highly-resistant individuals with ≥ 8 EPSPS gene copies) need to be mechanically removed before flowering from GR sugar beet fields. This research warrants the need to adopt ecologically based, multi-tactic strategies to reduce exposure of B. scoparia to glyphosate in GR sugar beet.

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Variable Inheritance of AmplifiedEPSPSGene Copies in Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

Weed Science ◽

10.1017/wsc.2018.65 ◽

2018 ◽

Vol 67 (2) ◽

pp. 176-182 ◽

Cited By ~ 6

Author(s):

Darci A. Giacomini ◽

Philip Westra ◽

Sarah M. Ward

Keyword(s):

Copy Number ◽

Single Gene ◽

Gene Copy Number ◽

Transgressive Segregation ◽

Palmer Amaranth ◽

Gene Copy ◽

Amaranthus Palmeri ◽

Gene Copies ◽

Central United States ◽

Free Environment

AbstractGlyphosate-resistant (GR) Palmer amaranth (Amaranthus palmeriS. Watson) is considered one of the most troublesome weeds in the southern and central United States, but results of previous research to determine the mode of inheritance of this trait have been conflicting and inconclusive. In this study, we examined segregation patterns ofEPSPSgene-copy numbers in F1and F2generations ofA. palmeriand found no evidence of a Mendelian single-gene pattern of inheritance. Transgressive segregation for copy number was exhibited by several F1and all of the F2families, most likely the product ofEPSPScopy-number variation within each plant. This variation was confirmed by assaying gene-copy number across clonal generations and among individual shoots on the same plant, demonstrating thatEPSPSamplification levels vary significantly within a single plant. Increases and decreases in copy number occurred in a controlled, stress-free environment in the absence of glyphosate, indicating thatEPSPSgene amplification is a random and variable process within the plant. The ability ofA. palmerito gain or loseEPSPSgene copies is a valuable adaptive trait, allowing this species to respond rapidly to selection pressures and changing environments.

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HER2 Gene Assessment in Liquid Biopsy of Gastric and Esophagogastric Junction Cancer Patients Qualified for Surgery

10.21203/rs.3.rs-60854/v1 ◽

2020 ◽

Author(s):

Anna Grenda ◽

Kamila Wojas-Krawczyk ◽

Tomasz Skoczylas ◽

Paweł Krawczyk ◽

Jadwiga Sierocińska-Sawa ◽

...

Keyword(s):

Cancer Cells ◽

Liquid Biopsy ◽

Copy Number ◽

Esophagogastric Junction ◽

Tumor Tissue ◽

Gene Copy Number ◽

Gene Copy ◽

Gene Copies ◽

Healthy Donors ◽

Esophagogastric Junction Cancer

Abstract Background Amplification of HER2 gene (ERBB2) and overexpression of HER2 protein on cancer cells are found in 10-26% of gastric cancer (GC) and esophagogastric junction cancer (EGJC). Gene copy number variation (CNV) could be detected in these patients in liquid biopsy and in cancer cells. Methods We analysed HER2 gene CNV used qPCR method in 87 sera collected from GC and EGJC patients before surgical treatment and in 40 sera obtained from healthy donors. HER2 gene CNV was also assessed in formalin-fixed paraffin-embedded (FFPE) tumor tissue. Furthermore, we assessed the number of HER2 gene copies and HER2 expression in cancer cells using the fluorescent in situ hybridization method (FISH) and immunohistochemistry (IHC). Results We found that the HER2 gene copy number in liquid biopsy was higher in GC and EGJC patients compared to healthy people (p=0.01). Moreover, EGJC patients had higher number of HER2 gene copies than healthy donors (p=0.0016). HER2 CNV examination could distinguish healthy individuals and patients with gastric or esophagogastric junction cancers with sensitivity and specificity of 58% and 98% (AUC=0.707, 95% CI: 0.593-0.821, p=0.004). We found that patients with a high copy number of the HER2 gene in the tumor tissue assessed by qPCR (but not by FISH) have significantly more often a high number of HER2 gene copies in liquid biopsy (p=0.04). Conclusions We suggested that HER2 testing in liquid biopsy could be used as an auxiliary method to analysis of HER2 status in tumor tissue in gastric or esophagogastric junction cancers.

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Confirmation of Glyphosate-Resistant Kochia (Kochia scoparia) from Sugar Beet Fields in Idaho and Oregon

Weed Technology ◽

10.1017/wet.2017.80 ◽

2017 ◽

Vol 32 (1) ◽

pp. 27-33 ◽

Cited By ~ 3

Author(s):

Vipan Kumar ◽

Joel Felix ◽

Don Morishita ◽

Prashant Jha

Keyword(s):

Sugar Beet ◽

Copy Number ◽

Cropping Systems ◽

Gene Copy Number ◽

Glyphosate Resistance ◽

Gene Copy ◽

Resistance Level ◽

Control Programs ◽

Gene Copies

AbstractGlyphosate-resistant (GR) kochia is an increasing management concern in major cropping systems of the northwestern US. In 2014, we investigated four putative GR kochia accessions (designated as ALA, VAL, WIL, DB) collected from sugar beet fields in eastern Oregon and southwestern Idaho to characterize the level of evolved glyphosate resistance and determine the relationship between the 5-enol-pyruvylshikimate-3-phospate synthase (EPSPS) gene copy number and level of glyphosate resistance. TheEPSPSgene copy number was used as a molecular marker to detect GR kochia in subsequent surveys in 2015 and 2016. Based on LD50values from a whole-plant dose-response study, the four putative GR kochia populations were 2.0- to 9.6-fold more resistant to glyphosate than the glyphosate-susceptible (GS) accession. In anin vivoleaf-disk shikimate assay, leaf disks of GS kochia plants treated with 100-μM glyphosate accumulated 2.4- to 4.0-fold higher amounts of shikimate than the GR plants. The four GR accessions had 2.7 to 9.1 relativeEPSPSgene copies compared with the GS accession (<1EPSPSgene copies), and there was a linear relationship betweenEPSPSgene copy number and glyphosate resistance level (LD50values). The 2015 and 2016 GR kochia survey results indicated that about half of the collected populations from sugar beet fields in eastern Oregon had developed resistance to glyphosate whereas only one population from the Idaho collection was confirmed glyphosate resistant. This is the first confirmation of GR kochia in sugar beet fields in eastern Oregon and southwestern Idaho. Diversified weed control programs will be required to prevent further development and spread of GR kochia in sugar beet-based rotations in this region.

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Intragenic and structural variation in the SMN locus and clinical variability in spinal muscular atrophy

Brain Communications ◽

10.1093/braincomms/fcaa075 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Renske I Wadman ◽

Marc D Jansen ◽

Marloes Stam ◽

Camiel A Wijngaarde ◽

Chantall A D Curial ◽

...

Keyword(s):

Spinal Muscular Atrophy ◽

Copy Number ◽

Muscular Atrophy ◽

Gene Copy Number ◽

Gene Copy ◽

Gene Copies ◽

Smn2 Gene ◽

Polymerase Chain ◽

Hybrid Genes ◽

Dependent Probe

Abstract Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1–4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1–SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.

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Determination of Cytochrome P450 2D6 (CYP2D6) Gene Copy Number by Real-Time Quantitative PCR

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb.2005.248 ◽

2005 ◽

Vol 2005 (3) ◽

pp. 248-253 ◽

Cited By ~ 40

Author(s):

Laurent Bodin ◽

Philippe H. Beaune ◽

Marie-Anne Loriot

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Copy Number ◽

Gene Dosage ◽

Gene Copy Number ◽

Rapid Identification ◽

Gene Copy ◽

Cyp2d6 Gene ◽

Real Time Quantitative Pcr ◽

Gene Copies

Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number). Using the2−ΔΔCtcalculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies), and 12 samples with duplicated genes. The average ratio ranged from1.02to1.28,1.85to2.21, and2.55to3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

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Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer's disease brains

eLife ◽

10.7554/elife.05116 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 64

Author(s):

Diane M Bushman ◽

Gwendolyn E Kaeser ◽

Benjamin Siddoway ◽

Jurgen W Westra ◽

Richard R Rivera ◽

...

Keyword(s):

Amyloid Precursor Protein ◽

Single Cell ◽

Dna Content ◽

Copy Number ◽

Gene Copy Number ◽

Precursor Protein ◽

Gene Copy ◽

Single Neurons ◽

Sporadic Alzheimer’S Disease ◽

App Gene

Previous reports have shown that individual neurons of the brain can display somatic genomic mosaicism of unknown function. In this study, we report altered genomic mosaicism in single, sporadic Alzheimer's disease (AD) neurons characterized by increases in DNA content and amyloid precursor protein (APP) gene copy number. AD cortical nuclei displayed large variability with average DNA content increases of ∼8% over non-diseased controls that were unrelated to trisomy 21. Two independent single-cell copy number analyses identified amplifications at the APP locus. The use of single-cell qPCR identified up to 12 copies of APP in sampled neurons. Peptide nucleic acid (PNA) probes targeting APP, combined with super-resolution microscopy detected primarily single fluorescent signals of variable intensity that paralleled single-cell qPCR analyses. These data identify somatic genomic changes in single neurons, affecting known and unknown loci, which are increased in sporadic AD, and further indicate functionality for genomic mosaicism in the CNS.

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From the sxtA4 Gene to Saxitoxin Production: What Controls the Variability Among Alexandrium minutum and Alexandrium pacificum Strains?

Frontiers in Microbiology ◽

10.3389/fmicb.2021.613199 ◽

2021 ◽

Vol 12 ◽

Author(s):

Solène Geffroy ◽

Marc-Marie Lechat ◽

Mickael Le Gac ◽

Georges-Augustin Rovillon ◽

Dominique Marie ◽

...

Keyword(s):

Gene Expression ◽

Genome Size ◽

Copy Number ◽

Gene Copy Number ◽

Toxin Production ◽

Gene Copy ◽

Alexandrium Minutum ◽

Copy Numbers ◽

Gene Copies ◽

Paralytic Shellfish

Paralytic shellfish poisoning (PSP) is a human foodborne syndrome caused by the consumption of shellfish that accumulate paralytic shellfish toxins (PSTs, saxitoxin group). In PST-producing dinoflagellates such as Alexandrium spp., toxin synthesis is encoded in the nuclear genome via a gene cluster (sxt). Toxin production is supposedly associated with the presence of a 4th domain in the sxtA gene (sxtA4), one of the core genes of the PST gene cluster. It is postulated that gene expression in dinoflagellates is partially constitutive, with both transcriptional and post-transcriptional processes potentially co-occurring. Therefore, gene structure and expression mode are two important features to explore in order to fully understand toxin production processes in dinoflagellates. In this study, we determined the intracellular toxin contents of twenty European Alexandrium minutum and Alexandrium pacificum strains that we compared with their genome size and sxtA4 gene copy numbers. We observed a significant correlation between the sxtA4 gene copy number and toxin content, as well as a moderate positive correlation between the sxtA4 gene copy number and genome size. The 18 toxic strains had several sxtA4 gene copies (9–187), whereas only one copy was found in the two observed non-toxin producing strains. Exploration of allelic frequencies and expression of sxtA4 mRNA in 11 A. minutum strains showed both a differential expression and specific allelic forms in the non-toxic strains compared with the toxic ones. Also, the toxic strains exhibited a polymorphic sxtA4 mRNA sequence between strains and between gene copies within strains. Finally, our study supported the hypothesis of a genetic determinism of toxin synthesis (i.e., the existence of several genetic isoforms of the sxtA4 gene and their copy numbers), and was also consistent with the hypothesis that constitutive gene expression and moderation by transcriptional and post-transcriptional regulation mechanisms are the cause of the observed variability in the production of toxins by A. minutum.

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