Spontaneous mutations in maize pollen are frequent in some lines and arise mainly from retrotranspositions and deletions

While studying spontaneous mutations at the maizebronze(bz) locus, we made the unexpected discovery that specific low-copy number retrotransposons are mobile in the pollen of some maize lines, but not of others. We conducted large-scale genetic experiments to isolate newbzmutations from severalBzstocks and recovered spontaneous stable mutations only in the pollen parent in reciprocal crosses. Most of the new stablebzmutations resulted from either insertions of low-copy number long terminal repeat (LTR) retrotransposons or deletions, the same two classes of mutations that predominated in a collection of spontaneouswxmutations [Wessler S (1997)The Mutants of Maize, pp 385–386]. Similar mutations were recovered at the closely linkedshlocus. These events occurred with a frequency of 2–4 × 10−5in two lines derived from W22 and in 4Co63, but not at all in B73 or Mo17, two inbreds widely represented in Corn Belt hybrids. Surprisingly, the mutagenic LTR retrotransposons differed in the active lines, suggesting differences in the autonomous element make-up of the lines studied. Some active retrotransposons, likeHopscotch,Magellan, andBs2, aBs1variant, were described previously; others, likeFotoandFocouin 4Co63, were not. By high-throughput sequencing of retrotransposon junctions, we established that retrotranposition ofHopscotch,Magellan, andBs2occurs genome-wide in the pollen of active lines, but not in the female germline or in somatic tissues. We discuss here the implications of these results, which shed light on the source, frequency, and nature of spontaneous mutations in maize.

Download Full-text

Phased Genotyping-by-Sequencing Enhances Analysis of Genetic Diversity and Reveals Divergent Copy Number Variants in Maize

G3 Genes|Genome|Genetics ◽

10.1534/g3.117.042036 ◽

2017 ◽

Vol 7 (7) ◽

pp. 2161-2170 ◽

Cited By ~ 9

Author(s):

Heather Manching ◽

Subhajit Sengupta ◽

Keith R Hopper ◽

Shawn W Polson ◽

Yuan Ji ◽

...

Keyword(s):

Copy Number ◽

High Throughput Sequencing ◽

Copy Number Variants ◽

Genotyping By Sequencing ◽

Genomic Libraries ◽

Reduced Representation ◽

Large Samples ◽

Snp Data ◽

Genome Wide ◽

Missing Data Problem

Abstract High-throughput sequencing (HTS) of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken from heterogeneous populations of heterozygous individuals. This requires that a number of issues encountered with GBS be considered, including the sequencing of nonoverlapping sets of loci across multiple GBS libraries, a common missing data problem that results in low call rates for markers per individual, and a tendency for applicability only in inbred line samples with sufficient linkage disequilibrium for accurate imputation. We addressed these issues while developing and validating a new, comprehensive platform for GBS. This study supports the notion that GBS can be tailored to particular aims, and using Zea mays our results indicate that large samples of unknown pedigree can be genotyped to obtain complete and accurate GBS data. Optimizing size selection to sequence a high proportion of shared loci among individuals in different libraries and using simple in silico filters, a GBS procedure was established that produces high call rates per marker (>85%) with accuracy exceeding 99.4%. Furthermore, by capitalizing on the sequence-read structure of GBS data (stacks of reads), a new tool for resolving local haplotypes and scoring phased genotypes was developed, a feature that is not available in many GBS pipelines. Using local haplotypes reduces the marker dimensionality of the genotype matrix while increasing the informativeness of the data. Phased GBS in maize also revealed the existence of reproducibly inaccurate (apparent accuracy) genotypes that were due to divergent copy number variants (CNVs) unobservable in the underlying single nucleotide polymorphism (SNP) data.

Download Full-text

CaSpER: Identification, visualization and integrative analysis of CNV events in multiscale resolution using single-cell or bulk RNA sequencing data

10.1101/426122 ◽

2018 ◽

Cited By ~ 1

Author(s):

Akdes Serin Harmancı ◽

Arif O. Harmanci ◽

Xiaobo Zhou

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Copy Number ◽

Large Scale ◽

Complete Characterization ◽

Integrative Analysis ◽

Sequencing Data ◽

Length Scales ◽

Expression Levels ◽

Genome Wide

AbstractRNA sequencing experiments generate large amounts of information about expression levels of genes. Although they are mainly used for quantifying expression levels, they contain much more biologically important information such as copy number variants (CNV). Here, we propose CaSpER, a signal processing approach for identification, visualization, and integrative analysis of focal and large-scale CNV events in multiscale resolution using either bulk or single-cell RNA sequencing data. CaSpER performs smoothing of the genome-wide RNA sequencing signal profiles in different multiscale resolutions, identifying CNV events at different length scales. CaSpER also employs a novel methodology for generation of genome-wide B-allele frequency (BAF) signal profile from the reads and utilizes it in multiscale fashion for correction of CNV calls. The shift in allelic signal is used to quantify the loss-of-heterozygosity (LOH) which is valuable for CNV identification. CaSpER uses Hidden Markov Models (HMM) to assign copy number states to regions. The multiscale nature of CaSpER enables comprehensive analysis of focal and large-scale CNVs and LOH segments. CaSpER performs well in accuracy compared to gold standard SNP genotyping arrays. In particular, analysis of single cell Glioblastoma (GBM) RNA sequencing data with CaSpER reveals novel mutually exclusive and co-occurring CNV sub-clones at different length scales. Moreover, CaSpER discovers gene expression signatures of CNV sub-clones, performs gene ontology (GO) enrichment analysis and identifies potential therapeutic targets for the sub-clones. CaSpER increases the utility of RNA-sequencing datasets and complements other tools for complete characterization and visualization of the genomic and transcriptomic landscape of single cell and bulk RNA sequencing data, especially in cancer research.

Download Full-text

Genome-Wide Analysis of Copy Number Analysis of Myelodysplastic Syndromes Using High-Density SNP-Genotyping Microarrays.

Blood ◽

10.1182/blood.v106.11.3420.3420 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3420-3420

Author(s):

Masashi Sanada ◽

Yasuhito Nannya ◽

Kumi Nakazaki ◽

Go Yamamoto ◽

Lili Wang ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Copy Number ◽

Large Scale ◽

Target Genes ◽

Chromosomal Abnormalities ◽

High Density ◽

Copy Number Alterations ◽

Genome Wide Analysis ◽

Conventional Cytogenetic Analysis ◽

Genome Wide

Abstract Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by impaired blood cell production due to ineffective hematopoiesis and high propensity to acute myeloid leukemias. One of the prominent features of MDS is the high frequency of unbalanced chromosomal abnormalities that result in genetic imbalances and copy number alterations. Although the chromosomal segments involved in these abnormalities are thought to contain relevant genes to the pathogenesis of MDS, conventional analyses including FISH have failed to identify critical regions small enough to pinpoint their target genes. Affymetrix® GeneChip® 100K/500K mapping arrays were originally developed for large-scale genotyping of more than 100,000/500,000 SNPs in two separate arrays, but the quantitative nature of the preparative whole-genome amplification and array hybridization thereafter also allows for accurate copy number estimate of the genome using these platforms at the resolutions of 21.3 kb and 5.4 kb with 116,204 and 520,000 oligonucleotide probes, respectively. Here we developed robust algorithms (CNAG) for copy number detection using 100K and/or 500K arrays and analyzed 88 MDS samples on these platforms in order to identify relevant genes for development of MDS. With these huge numbers of uniformly distributed SNP probes, numerous copy number alterations were sensitively detected in cases with MDS with more numbers of abnormalities found in advanced diseases (RAEB and RAEB-t). In addition to large-scale alterations of various chromosomal segments previously reported in these syndromes, a number of small cryptic chromosomal abnormalities were identified that would escape conventional cytogenetic analysis or array CGH analysis. Minimum overlapping deletions in 5q, 7q, 12p, 13q, and 20q were precisely defined, although no pinpoint homozygous deletions were detected within these regions. A common 20q deletion spans a 400 kb segment harboring five transcriptomes and the common 12p deletion defines a 1.3 Mb region that contains the ETV6 gene. Other common overlapping abnormalities include deletions in 21q22, 17q13, and gains of 11q25. Genome-wide analysis of copy number changes using high-density oligonucleotide arrays provides valuable information about genetic abnormalities in MDS.

Download Full-text

Dynamics of R1 and R2 Elements in the rDNA Locus of Drosophila simulans

Genetics ◽

10.1093/genetics/158.4.1557 ◽

2001 ◽

Vol 158 (4) ◽

pp. 1557-1567 ◽

Cited By ~ 3

Author(s):

César E Pérez-González ◽

Thomas H Eickbush

Keyword(s):

Copy Number ◽

Rdna Locus ◽

Drosophila Simulans ◽

Rrna Gene ◽

Ltr Retrotransposons ◽

Pcr Assay ◽

X Chromosomes ◽

Low Copy Number ◽

The Many ◽

The Individual

Abstract The mobile elements R1 and R2 insert specifically into the rRNA gene locus (rDNA locus) of arthropods, a locus known to undergo concerted evolution, the recombinational processes that preserve the sequence homogeneity of all repeats. To monitor how rapidly individual R1 and R2 insertions are turned over in the rDNA locus by these processes, we have taken advantage of the many 5′ truncation variants that are generated during the target-primed reverse transcription mechanism used by these non-LTR retrotransposons for their integration. A simple PCR assay was designed to reveal the pattern of the 5′ variants present in the rDNA loci of individual X chromosomes in a population of Drosophila simulans. Each rDNA locus in this population was found to have a large, unique collection of 5′ variants. Each variant was present at low copy number, usually one copy per chromosome, and was seldom distributed to other chromosomes in the population. The failure of these variants to spread to other units in the same rDNA locus suggests a strong recombinational bias against R1 and R2 that results in the individual copies of these elements being rapidly lost from the rDNA locus. This bias suggests a significantly higher frequency of R1 and R2 retrotransposition than we have previously suggested.

Download Full-text

Gene Silencing Triggered by Non-LTR Retrotransposons in the Female Germline ofDrosophila melanogaster

Genetics ◽

10.1093/genetics/164.2.521 ◽

2003 ◽

Vol 164 (2) ◽

pp. 521-531 ◽

Cited By ~ 1

Author(s):

Stéphanie Robin ◽

Séverine Chambeyron ◽

Alain Bucheton ◽

Isabelle Busseau

Keyword(s):

Drosophila Melanogaster ◽

Transposable Elements ◽

Gene Silencing ◽

Bombyx Mori ◽

Copy Number ◽

Transcription Unit ◽

Ltr Retrotransposons ◽

Endogenous Gene ◽

Sequence Identity ◽

Female Germline

AbstractSeveral studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent genesilencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing.

Download Full-text

Canvas: versatile and scalable detection of copy number variants

10.1101/036194 ◽

2016 ◽

Author(s):

Eric Roller ◽

Sergii Ivakhno ◽

Steve Lee ◽

Thomas Royce ◽

Stephen Tanner

Keyword(s):

Copy Number ◽

Large Scale ◽

Copy Number Variants ◽

Variant Calling ◽

Experimental Designs ◽

Genome Wide ◽

Whole Exome ◽

Sequencing Studies ◽

Copy Number Changes ◽

Robust Variant

Motivation: Increased throughput and diverse experimental designs of large-scale sequencing studies necessi-tate versatile, scalable and robust variant calling tools. In particular, identification of copy number changes re-mains a challenging task due to their complexity, susceptibility to sequencing biases, variation in coverage data and dependence on genome-wide sample properties, such as tumor polyploidy or polyclonality in cancer samples. Results: We have developed a new tool, Canvas, for identification of copy number changes from diverse se-quencing experiments including whole-genome matched tumor-normal and single-sample normal re-sequencing, as well as whole-exome matched and unmatched tumor-normal studies. In addition to variant calling, Canvas infers genome-wide parameters such as cancer ploidy, purity and heterogeneity. It provides fast and simple to execute workflows that can scale to thousands of samples and can be easily incorporated into existing variant calling pipelines. Availability: Canvas is distributed under an open source license and can be downloaded from https://github.com/Illumina/canvas.

Download Full-text

CNest: A Novel Copy Number Association Discovery Method Uncovers 862 New Associations from 200,629 Whole Exome Sequence Datasets in the UK Biobank

10.1101/2021.08.19.456963 ◽

2021 ◽

Author(s):

Tomas W Fitzgerald ◽

Ewan Birney

Keyword(s):

Copy Number ◽

Large Scale ◽

Association Studies ◽

Genomic Variation ◽

Next Generation Sequencing Data ◽

Genome Wide Association Studies ◽

Uk Biobank ◽

Genome Wide ◽

The Uk ◽

Ngs Data

Copy number variation (CNV) has long been known to influence human traits having a rich history of research into common and rare genetic disease and although CNV is accepted as an important class of genomic variation, progress on copy number (CN) phenotype associations from Next Generation Sequencing data (NGS) has been limited, in part, due to the relative difficulty in CNV detection and an enrichment for large numbers of false positives. To date most successful CN genome wide association studies (CN-GWAS) have focused on using predictive measures of dosage intolerance or gene burden tests to gain sufficient power for detecting CN effects. Here we present a novel method for large scale CN analysis from NGS data generating robust CN estimates and allowing CN-GWAS to be performed genome wide in discovery mode. We provide a detailed analysis in the large scale UK BioBank resource and a specifically designed software package for deriving CN estimates from NGS data that are robust enough to be used for CN-GWAS. We use these methods to perform genome wide CN-GWAS analysis across 78 human traits discovering 862 genetic associations that are likely to contribute strongly to trait distributions based solely on their CN or by acting in concert with other genetic variation. Finally, we undertake an analysis comparing CNV and SNP association signals across the same traits and samples, defining specific CNV association classes based on whether they could be detected using standard SNP-GWAS in the UK Biobank.

Download Full-text

Comparative whole plastome and low copy number phylogenetics of the core Saccharinae and Sorghinae.

10.1101/2022.01.04.474895 ◽

2022 ◽

Author(s):

Dyfed Lloyd Evans ◽

Ben Hughes ◽

Shailesh Vinay Joshi

Keyword(s):

Copy Number ◽

Large Scale ◽

New World ◽

Genetic Resource ◽

Phylogenetic Analyses ◽

World Species ◽

Separate Genus ◽

The Core ◽

Low Copy Number ◽

Long Read

Despite over 60 years' worth of taxonomic efforts, the relationships between sugarcane (Saccharum hybrid cultivars), Sorghum and their closest evolutionary relatives remain largely unresolved. Even relationships between generally accepted genera such as Miscanthus and Saccharum have not been examined in any large-scale molecular detail. Genera such as Erianthus, Miscanthidium and Narenga pose even greater taxonomic contention. Erianthus is not monophyletic and Erianthus sect. Ripidium (Valdés and Scholz 2006, Lloyd Evans et al. 2019a; Welker et al. 2019) represents a distinct and separate genus, Tripidium Scholz. Miscanthidium is placed within Miscanthus by many workers, whilst the New World Erianthus species and Narenga are currently placed within Saccharum. As these species represent a significant portion of the gene pool that sugarcane breeders use for introgression into sugarcane, their taxonomic placement and relationships to Saccharum are of significant economic import. Erianthus species from the Americas have not been significantly employed in sugarcane breeding and may represent an untapped genetic resource. In an attempt to resolve the taxonomic relationships of these genera, we have assembled three novel chloroplasts, from Miscanthidium capense, Miscanthidium junceum and Narenga porphyrocoma (this latter assembled from transcriptomic and long read data). In parallel, five low copy number loci have been assembled from species within Saccharum, Miscanthus, Sarga and Sorghum. Phylogenetic analyses were performed using both low copy number genes and whole chloroplasts. The phylogenetic results were compared with karyotype data to circumscribe the genera most closely related to sugarcane. We reveal that genera Miscanthus and Saccharum are monophyletic and have never undergone polyploidization outside their own genera. Genera Erianthus, Miscanthidium and Narenga are allopolyploids, which excludes them from being members of Saccharum and Miscanthus. Moreover, all three of these genera have divergent evolutionary histories. We therefore support the use of the genera Miscanthus, Miscanthidium, Erianthus (for the New World Species) and Narenga for those species and genera most closely allied to Saccharum. Our data demonstrate that all these genera should be excluded from Saccharum sensu lato.

Download Full-text

Array-Based Comparative Genomic Hybridization for Genome-Wide Analysis of DNA Copy Number in Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v104.11.3420.3420 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3420-3420

Author(s):

Masashi Sanada ◽

Yasuhito Nanya ◽

Akira Hangaishi ◽

Noriko Hosoya ◽

LiLi Wang ◽

...

Keyword(s):

Human Genome ◽

Tumor Cells ◽

Copy Number ◽

Large Scale ◽

Array Cgh ◽

Chromosomal Abnormalities ◽

Comparative Genomic ◽

Normal Cells ◽

Genome Wide Analysis ◽

Genome Wide

Abstract Myelodysplastic syndrome(MDS)is a clonal disorder of hematopoietic stem cells characterized by ineffective hematopoiesis and propensity to acute myeloid leukemias. The conversion of a normal stem cell into a preleukemic and ultimately leukemic state is thought to be a multistep process requiring accumulation of a number of genetic changes. Conventional cytogenetic analysis has disclosed a number of chromosome abnormalities common to MDS and provided valuable clues to characterize these genetic lesions, rarity of balanced translocations and relative predominance of unbalanced abnormalities in MDS, including gene deletions and amplifications. However conventional analytical methods provide only limited resolutions of analysis for identification of genetic gains and losses and prevent further molecular delineation of relevant genes to the pathogenesis of MDS.</PRE> Array-based comparative genomic hybridization (CGH) is a robust technique to enable rapid and comprehensive genome-wide analysis of genetic aberrations in cancers, in which differentially labeled DNAs from both tumor and normal samples are comparatively hybridized to a large number of genomic DNAs. In this study, we constructed a high-quality array-based CGH system for genome-wide analysis of chromosomal abnormalities to identify candidate target genes of MDS. Our whole genome arrays consisted of 3,300 BAC/PAC clones, thus having an average resolution of 1.0 Mb over the whole human genome. Each clone was amplified with degenerated oligonucleotide primed-PCR (DOP-PCR) and the amplified products were spotted in duplicate grids onto aminosilan-coated glass slides. For more high-resolution analysis, we employed the GeneChip Mapping 100k arrays (Affymetrix), originally developed for large-scale SNP typing, as a tool for detection of copy number changes in selected MDS cases. It contains 116,204 different SNPs on two separate arrays, covering the whole human genome with an average resolution of 21 kb. With this arrays DNA copy number’s changes could be estimated by comparing intensity of SNP signals of tumor cells with that of normal cells from the same patients. In addition, using paired samples from tumor cells and normal cells, large-scale LOH analysis became also possible.</PRE> In total, 54 MDS samples were analyzed using our array CGH system. In addition to large chromosomal changes, including loss of 5q, 7q, 13q, and 20q, and gain of the whole chromosome 8, a number of small, cryptic chromosomal abnormalities were identified that would escape from conventional cytogenetic detection. Many of these abnormalities were represented only by a single PAC/BAC clone. In several chromosome regions, including 3q13, 5p15, 13p33, and 20q12, there existed commonly deleted regions, which could be confirmed by FISH analysis. Similarly gains of genetic materials were found on 8p23 and 17p13. Several genes were identified within these regions that may be candidates for relevant genes to these genetic alterations. In conclusion, genome-profiling using array CGH techniques were highly useful tools for delineating the pathogenesis of MDS.</PRE>

Download Full-text

High-Throughput Sequencing of the Human Platelet Transcriptome

Blood ◽

10.1182/blood.v116.21.481.481 ◽

2010 ◽

Vol 116 (21) ◽

pp. 481-481

Author(s):

Paul F Bray ◽

Paolo M. Fortina ◽

Srikanth Nagalla ◽

Kathleen Delgrosso ◽

Adam Ertel ◽

...

Keyword(s):

High Throughput ◽

Copy Number ◽

High Throughput Sequencing ◽

Human Platelet ◽

Relative Reduction ◽

Genome Wide Association Studies ◽

High Background ◽

Genome Wide ◽

Typical Sequence ◽

Local Sequence

Abstract Abstract 481 Most successful DNA-based genome wide association studies identify genomic regions, not genes themselves, and the findings are often devoid of context or mechanism. To identify the genetic basis of disease and disease traits, it is imperative to characterize the quantity and forms of the genes that are expressed in the tissue of interest. It is not feasible to use primary megakaryocytes to profile mRNA from large numbers of subjects, but platelet RNA is easy to obtain. Others and we have previously surveyed genome-wide platelet RNA expression using microarrays, an approach that has had a major impact on systems biology. However, microarrays have a number of limitations, including the use of probes only to known transcripts, a limited dynamic range for quantifying very low and high levels of transcripts, high background levels from cross-hybridization, and complicated normalization schemes to compare expression levels across experiments. Novel high-throughput sequencing approaches that overcome the limitations of microarrays have recently become available. RNA sequencing (RNAseq) has a remarkable ability to quantify mRNAs and provide information about transcript sequence variations, including single nucleotide changes and alternately spliced exons. The goal of these studies was to apply RNAseq to capture platelet transcriptome complexity. Total RNA was prepared using leukocyte-depleted platelets (LDP; less than 1 WBC per 5 million platelets) from 4 donors; 2 were studied twice each. Analysis of this material showed that compared to nucleated cells (HeLa, Meg-01), platelets had 50%-90% less ribosomal RNA, and high levels of messenger and small RNAs (Agilent 2100). The major reduction in platelet rRNA was confirmed by RNA gel analysis. The platelet whole transcriptomes were analyzed via the Applied Biosystems (AB) SOLiD 3Plus next generation sequencing protocols and platform. A typical sequence run generated ∼250 million reads of 50 bp each. We observed more than 30,000 independent platelet mRNA-coding transcripts from about 10,000 genes, demonstrating substantial numbers of variant isoforms. The increased sensitivity of RNAseq for low copy number is clear from these results, because prior platelet transcriptome studies using microarrays have identified only 1500–6000 expressed genes. As an example, the platelet-specific transcript, ITGA2B, showed very high copy number in platelets, but no expression in HeLa cells and modest expression in the megakaryocyte cell line, Meg-01. As is expected for RNA-Seq data, the density of mapped reads varies by exon and local sequence. We also provide examples of newly discovered SNPs that encode non-conservative amino acid changes (AKT2 1209A/T; PIK3CB 837C/G) and alter consensus exon/intron splice junction sites (P2YR12 nt 65 G/A). We have also identified a major difference in the ratio of two splice variants of the FcRg chain, 4:1 in one human platelet donor and 49:1 in another. In summary, we have demonstrated that RNAseq can accurately and sensitively determine the quantity and quality of variations in individual platelet transcriptomes. It appears that the the platelet transcriptome is approximately 10 times more complex than previously thought. The major relative reduction in platelet rRNA may be an advantage for characterizing functional platelet transcripts. RNAseq should permit better understanding of the molecular mechanisms regulating platelet physiology and identify novel genetic variants that contribute to disorders of thrombosis and hemostasis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text