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Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing.

ABSTRACT: Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study, we establish a low-coverage whole-genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar 'Novaria' and found that it harbours multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deletions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar 'Williams'. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy-treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.

SUBMITTER: Datta S

PROVIDER: S-EPMC6097122 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing.

Datta Sneha S Jankowicz-Cieslak Joanna J Nielen Stephan S Ingelbrecht Ivan I Till Bradley J BJ

Plant biotechnology journal 20180224

Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical muta ...[more]

PMID: 29476650

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Project description:BackgroundWhole genome sequencing enables a high resolution view of the human genome and provides unique insights into genome structure at an unprecedented scale. There have been a number of tools to infer copy number variation in the genome. These tools, while validated, also include a number of parameters that are configurable to genome data being analyzed. These algorithms allow for normalization to account for individual and population-specific effects on individual genome CNV estimates but the impact of these changes on the estimated CNVs is not well characterized. We evaluate in detail the effect of normalization methodologies in two CNV algorithms FREEC and CNV-seq using whole genome sequencing data from 8 individuals spanning four populations.MethodsWe apply FREEC and CNV-seq to a sequencing data set consisting of 8 genomes. We use multiple configurations corresponding to different read-count normalization methodologies in FREEC, and statistically characterize the concordance of the CNV calls between FREEC configurations and the analogous output from CNV-seq. The normalization methodologies evaluated in FREEC are: GC content, mappability and control genome. We further stratify the concordance analysis within genic, non-genic, and a collection of validated variant regions.ResultsThe GC content normalization methodology generates the highest number of altered copy number regions. Both mappability and control genome normalization reduce the total number and length of copy number regions. Mappability normalization yields Jaccard indices in the 0.07 - 0.3 range, whereas using a control genome normalization yields Jaccard index values around 0.4 with normalization based on GC content. The most critical impact of using mappability as a normalization factor is substantial reduction of deletion CNV calls. The output of another method based on control genome normalization, CNV-seq, resulted in comparable CNV call profiles, and substantial agreement in variable gene and CNV region calls.ConclusionsChoice of read-count normalization methodology has a substantial effect on CNV calls and the use of genomic mappability or an appropriately chosen control genome can optimize the output of CNV analysis.

Project description:BackgroundNon-invasive, especially the urine-based diagnosis of prostate cancer (PCa) remains challenging. Although prostate cancer antigen (PSA) is widely used in prostate cancer screening, the false positives may result in unnecessary invasive procedures. PSA elevated patients are triaged to further evaluation of free/total PSA ratio (f/t PSA), to find out potential clinically significant PCa before undergoing invasive procedures. Genomic instability, especially chromosomal copy number variations (CNVs) were proved much more tumor specific. Here we performed a prospective study to evaluate the diagnostic value of CNV via urine-exfoliated cell DNA analysis in PCa.MethodsWe enrolled 28 PSA elevated patients (≥ 4 ng/ml), including 16 PCa, 9 benign prostate hypertrophy (BPH) and 3 prostatic intraepithelial neoplasia (PIN). Fresh initial portion urine was collected after hospital admission. Urine exfoliated cell DNA was analyzed by low coverage Whole Genome Sequencing, followed by CNV genotyping by the prostate cancer chromosomal aneuploidy detector (ProCAD). CNVs were quantified in absolute z-score (|Z|). Serum free/total PSA ratio (f/t PSA) was reported altogether.ResultsIn patients with PCa, the most frequent CNV events were chr3q gain (n = 2), chr8q gain (n = 2), chr2q loss (n = 4), and chr18q loss (n = 3). CNVs were found in 81.2% (95% Confidence Interval (CI) 53.7-95.0%) PCa. No CNV was identified in BPH patients. A diagnosis model was established by incorporating all CNVs. At the optimal cutoff of |Z|≥ 2.50, the model reached an AUC of 0.91 (95% CI 0.83-0.99), a sensitivity of 81.2% and a specificity of 100%. The CNV approach significantly outperformed f/t PSA (AUC = 0.62, P = 0.012). Further analyses showed that the CNV positive rate was significantly correlated with tumor grade. CNVs were found in 90.9% (95% CI 57.1-99.5%) high grade tumors and 60.0% (95% CI 17.0-92.7%) low grade tumors. No statistical significance was found for patient age, BMI, disease history and family history.ConclusionsUrine exfoliated cells harbor enriched CNV features in PCa patients. Urine detection of CNV might be a biomarker for PCa diagnosis, especially in terms of the clinically significant high-grade tumors.

Dataset Information

Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing.

Publications

Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing.

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