Copy number variant detection with low-coverage whole-genome sequencing is a viable replacement for the traditional array-CGH

Copy number variations (CNVs) are a type of structural variant involving alterations in the number of copies of specific regions of DNA, which can either be deleted or duplicated. CNVs contribute substantially to normal population variability; however, abnormal CNVs cause numerous genetic disorders. Nowadays, several methods for CNV detection are used, from the conventional cytogenetic analysis through microarray-based methods (aCGH) to next-generation sequencing (NGS). We present GenomeScreen - NGS based CNV detection method based on a previously described CNV detection algorithm used for non-invasive prenatal testing (NIPT). We determined theoretical limits of its accuracy and confirmed it with extensive in-silico study and already genotyped samples. Theoretically, at least 6M uniquely mapped reads are required to detect CNV with a length of 100 kilobases (kb) or more with high confidence (Z-score > 7). In practice, the in-silico analysis showed the requirement at least 8M to obtain >99% accuracy (for 100 kb deviations). We compared GenomeScreen with one of the currently used aCGH methods in diagnostic laboratories, which has a 200 kb mean resolution. GenomeScreen and aCGH both detected 59 deviations, GenomeScreen furthermore detected 134 other (usually) smaller variations. Furthermore, the overall cost per sample is about 2-3x lower in the case of GenomeScreen.

Download Full-text

Copy Number Variant Detection with Low-Coverage Whole-Genome Sequencing Represents a Viable Alternative to the Conventional Array-CGH

Diagnostics ◽

10.3390/diagnostics11040708 ◽

2021 ◽

Vol 11 (4) ◽

pp. 708

Author(s):

Marcel Kucharík ◽

Jaroslav Budiš ◽

Michaela Hýblová ◽

Gabriel Minárik ◽

Tomáš Szemes

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

In Silico ◽

Copy Number ◽

Normal Population ◽

Copy Number Variations ◽

Whole Genome ◽

Real Patient ◽

Low Coverage ◽

Cnv Detection

Copy number variations (CNVs) represent a type of structural variant involving alterations in the number of copies of specific regions of DNA that can either be deleted or duplicated. CNVs contribute substantially to normal population variability, however, abnormal CNVs cause numerous genetic disorders. At present, several methods for CNV detection are applied, ranging from the conventional cytogenetic analysis, through microarray-based methods (aCGH), to next-generation sequencing (NGS). In this paper, we present GenomeScreen, an NGS-based CNV detection method for low-coverage, whole-genome sequencing. We determined the theoretical limits of its accuracy and obtained confirmation in an extensive in silico study and in real patient samples with known genotypes. In theory, at least 6 M uniquely mapped reads are required to detect a CNV with the length of 100 kilobases (kb) or more with high confidence (Z-score > 7). In practice, the in silico analysis required at least 8 M to obtain >99% accuracy (for 100 kb deviations). We compared GenomeScreen with one of the currently used aCGH methods in diagnostic laboratories, which has mean resolution of 200 kb. GenomeScreen and aCGH both detected 59 deviations, while GenomeScreen furthermore detected 134 other (usually) smaller variations. When compared to aCGH, overall performance of the proposed GenemoScreen tool is comparable or superior in terms of accuracy, turn-around time, and cost-effectiveness, thus providing reasonable benefits, particularly in a prenatal diagnosis setting.

Download Full-text

Optical Genome Mapping as a Next-Generation Cytogenomic Tool for Detection of Structural and Copy Number Variations for Prenatal Genomic Analyses

Genes ◽

10.3390/genes12030398 ◽

2021 ◽

Vol 12 (3) ◽

pp. 398

Author(s):

Nikhil Shri Sahajpal ◽

Hayk Barseghyan ◽

Ravindra Kolhe ◽

Alex Hastie ◽

Alka Chaubey

Keyword(s):

Copy Number ◽

Southern Blotting ◽

Genome Mapping ◽

Genetic Disorders ◽

Prenatal Testing ◽

Standard Of Care ◽

Copy Number Variations ◽

Repeat Expansion ◽

Chromosomal Microarray ◽

Next Generation

Global medical associations (ACOG, ISUOG, ACMG) recommend diagnostic prenatal testing for the detection and prevention of genetic disorders. Historically, cytogenetic methods such as karyotype analysis, fluorescent in situ hybridization (FISH) and chromosomal microarray (CMA) are utilized worldwide to diagnose common syndromes. However, the limitations of each of these methods, either performed in tandem or simultaneously, demonstrates the need of a revolutionary technology that can alleviate the need for multiple technologies. Optical genome mapping (OGM) is a novel method that fills this void by being able to detect all classes of structural variations (SVs), including copy number variations (CNVs). OGM is being adopted by laboratories as a tool for both postnatal constitutional genetic disorders and hematological malignancies. This commentary highlights the potential for OGM to become a standard of care in prenatal genetic testing based on its capability to comprehensively identify large balanced and unbalanced SVs (currently the strength of karyotyping and metaphase FISH), CNVs (by CMA), repeat contraction disorders (by Southern blotting) and multiple repeat expansion disorders (by PCR-based methods or Southern blotting). Next-generation sequencing (NGS) methods are excellent at detecting sequence variants, but they are unable to accurately resolve repeat regions of the genome, which limits their ability to detect all classes of SVs. Notably, multiple molecular methods are used to identify repeat expansion and contraction disorders in routine clinical laboratories around the world. With non-invasive prenatal testing (NIPT) becoming the standard of care screening assay for all global pregnancies, we anticipate that OGM can provide a high-resolution, cytogenomic assay to be employed following a positive NIPT screen or for high-risk pregnancies with an abnormal ultrasound. Accurate detection of all types of genetic disorders by OGM, such as liveborn aneuploidies, sex chromosome anomalies, microdeletion/microduplication syndromes, repeat expansion/contraction disorders is key to reducing the global burden of genetic disorders.

Download Full-text

Validation of Copy Number Variants Detection from Pregnant Plasma Using Low-Pass Whole-Genome Sequencing in Noninvasive Prenatal Testing-Like Settings

Diagnostics ◽

10.3390/diagnostics10080569 ◽

2020 ◽

Vol 10 (8) ◽

pp. 569

Author(s):

Michaela Hyblova ◽

Maria Harsanyova ◽

Diana Nikulenkov-Grochova ◽

Jitka Kadlecova ◽

Marcel Kucharik ◽

...

Keyword(s):

Copy Number ◽

Copy Number Variants ◽

Prenatal Testing ◽

Detection Algorithm ◽

Detection Sensitivity ◽

Clinical Settings ◽

Noninvasive Prenatal Testing ◽

Low Pass ◽

Cnv Detection ◽

Fetal Fraction

Detection of copy number variants as an integral part of noninvasive prenatal testing is increasingly used in clinical practice worldwide. We performed validation on plasma samples from 34 pregnant women with known aberrations using cell-free DNA sequencing to evaluate the sensitivity for copy number variants (CNV) detection using an in-house CNV fraction-based detection algorithm. The sensitivity for CNVs smaller than 3 megabases (Mb), larger than 3Mb, and overall was 78.57%, 100%, and 90.6%, respectively. Regarding the fetal fraction, detection sensitivity in the group with a fetal fraction of less than 10% was 57.14%, whereas there was 100% sensitivity in the group with fetal fraction exceeding 10%. The assay is also capable of indicating whether the origin of an aberration is exclusively fetal or fetomaternal/maternal. This validation demonstrated that a CNV fraction-based algorithm was applicable and feasible in clinical settings as a supplement to testing for common trisomies 21, 18, and 13.

Download Full-text

The pattern of gene copy number variations (CNVs) in hepatocellular carcinoma; in silico analysis

10.21203/rs.3.rs-20514/v1 ◽

2020 ◽

Author(s):

Hossein Ansari ◽

Arman Shahrisa ◽

Maryam Tahmaseby ◽

Zahra Mohammadi ◽

Vinicio Carloni ◽

...

Keyword(s):

Tumor Suppressor ◽

In Silico ◽

Copy Number ◽

In Silico Analysis ◽

Multivesicular Body ◽

Copy Number Variations ◽

Molecular Networks ◽

Gene Copy ◽

Body Protein ◽

Silico Analysis

Abstract Cancer-associated death is the second leading cause of death worldwide. Study of the involved molecular networks of cancers can identify the potential target for early diagnosis, efficient therapies, and predictive prognosis of patients with cancer. Copy number variations are one type of DNA mutations which has been connected with human cancers. The CNVs can be used to find the regions of the genome involved in cancer phenotypes. This study is aimed to perform genome-wide chromosomal CNVs in HCC samples to find hotspot regions of disease using in silico analysis. The obtained data showed that gain of chromosome 1q and loss of 8p were frequently observed in target cancerous tissues. All the gains and losses were associated with tumor grade and metastasis. However, the amplification of YY1AP1 (Yin Yang-1 Associated Protein 1) and deletion of CHMP7 (Charged Multivesicular Body Protein 7) were observed in most of patients. These expression levels of YY1AP1 and CHMP7 were also upregulated and downregulated in cancerous samples respectively. Additionally, these two genes interact with critical oncogene and tumor suppressor genes like MDM2 (Mouse double minute 2 homolog) and VHL (von Hippel-Lindau tumor suppressor) showing the potential of these genes in HCC pathogenesis. Based on the observed data we suggest the 1q and 8p as candidate regions for HCC for researches especially YY1AP1 and CHMP7 loci.

Download Full-text

Expanding the Scope of Non-invasive Prenatal Testing to Detect Fetal Chromosomal Copy Number Variations

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.649169 ◽

2021 ◽

Vol 8 ◽

Author(s):

Songchang Chen ◽

Lanlan Zhang ◽

Jiong Gao ◽

Shuyuan Li ◽

Chunxin Chang ◽

...

Keyword(s):

Copy Number ◽

Detection Rate ◽

Prenatal Testing ◽

Read Depth ◽

Copy Number Variations ◽

Chromosomal Microarray ◽

Chromosomal Microarray Analysis ◽

Non Invasive ◽

Positive Detection Rate ◽

Cnv Detection

Non-invasive prenatal testing (NIPT) for common fetal trisomies is effective. However, the usefulness of cell-free DNA testing to detect other chromosomal abnormalities is poorly understood. We analyzed the positive rate at different read depths in next-generation sequencing (NGS) and identified a strategy for fetal copy number variant (CNV) detection in NIPT. Pregnant women who underwent NIPT by NGS at read depths of 4–6 M and fetuses with suspected CNVs were analyzed by amniocentesis and chromosomal microarray analysis (CMA). These fetus samples were re-sequenced at a read depth of 25 M and the positive detection rate was determined. With the increase in read depth, the positive CNV detection rate increased. The positive CNV detection rates at 25 M with small fragments were higher by NGS than by karyotype analysis. Increasing read depth in NGS improves the positive CNV detection rate while lowering the false positive detection rate. NIPT by NGS may be an accurate method of fetal chromosome analysis and reduce the rate of birth defects.

Download Full-text

Performance of In Silico Prediction Tools for the Detection of Germline Copy Number Variations in Cancer Predisposition Genes in 4208 Female Index Patients with Familial Breast and Ovarian Cancer

Cancers ◽

10.3390/cancers13010118 ◽

2021 ◽

Vol 13 (1) ◽

pp. 118

Author(s):

Louisa Lepkes ◽

Mohamad Kayali ◽

Britta Blümcke ◽

Jonas Weber ◽

Malwina Suszynska ◽

...

Keyword(s):

Ovarian Cancer ◽

In Silico ◽

Copy Number ◽

Gc Content ◽

Copy Number Variations ◽

Cancer Predisposition ◽

Predictive Values ◽

Prediction Tools ◽

Predisposition Genes ◽

Female Index

The identification of germline copy number variants (CNVs) by targeted next-generation sequencing (NGS) frequently relies on in silico CNV prediction tools with unknown sensitivities. We investigated the performances of four in silico CNV prediction tools, including one commercial (Sophia Genetics DDM) and three non-commercial tools (ExomeDepth, GATK gCNV, panelcn.MOPS) in 17 cancer predisposition genes in 4208 female index patients with familial breast and/or ovarian cancer (BC/OC). CNV predictions were verified via multiplex ligation-dependent probe amplification. We identified 77 CNVs in 76 out of 4208 patients (1.81%); 33 CNVs were identified in genes other than BRCA1/2, mostly in ATM, CHEK2, and RAD51C and less frequently in BARD1, MLH1, MSH2, PALB2, PMS2, RAD51D, and TP53. The Sophia Genetics DDM software showed the highest sensitivity; six CNVs were missed by at least one of the non-commercial tools. The positive predictive values ranged from 5.9% (74/1249) for panelcn.MOPS to 79.1% (72/91) for ExomeDepth. Verification of in silico predicted CNVs is required due to high frequencies of false positive predictions, particularly affecting target regions at the extremes of the GC content or target length distributions. CNV detection should not be restricted to BRCA1/2 due to the relevant proportion of CNVs in further BC/OC predisposition genes.

Download Full-text

Expanded noninvasive prenatal testing for fetal aneuploidy and copy number variations and parental willingness for invasive diagnosis in a cohort of 18,516 cases

BMC Medical Genomics ◽

10.1186/s12920-021-00955-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yunsheng Ge ◽

Jia Li ◽

Jianlong Zhuang ◽

Jian Zhang ◽

Yanru Huang ◽

...

Keyword(s):

Prenatal Diagnosis ◽

High Risk ◽

Pregnant Women ◽

Copy Number ◽

Prenatal Testing ◽

Copy Number Variations ◽

Trisomy 13 ◽

Noninvasive Prenatal Testing ◽

Predictive Values ◽

Parental Willingness

Abstract Background Noninvasive prenatal testing (NIPT) has been wildly used to screen for common aneuplodies. In recent years, the test has been expanded to detect rare autosomal aneuploidies (RATs) and copy number variations (CNVs). This study was performed to investigate the performance of expanded noninvasive prenatal testing (expanded NIPT) in screening for common trisomies, sex chromosomal aneuploidies (SCAs), rare autosomal aneuploidies (RATs), and copy number variations (CNVs) and parental willingness for invasive prenatal diagnosis in a Chinese prenatal diagnosis center. Methods A total of 24,702 pregnant women were retrospectively analyzed at the Women and Children’s Hospital from January 2013 to April 2019, among which expanded NIPT had been successfully conducted in 24,702 pregnant women. The high-risk expanded NIPT results were validated by karyotype analysis and chromosomal microarray analysis. All the tested pregnant women were followed up for pregnancy outcomes. Results Of the 24,702 cases, successful follow-up was conducted in 98.77% (401/446) of cases with common trisomies and SCAs, 91.95% (80/87) of RAT and CNV cases, and 76.25% (18,429/24,169) of cases with low-risk screening results. The sensitivity of expanded NIPT was 100% (95% confidence interval[CI], 97.38–100%), 96.67%(95%CI, 82.78–99.92%), and 100%(95%CI, 66.37–100.00%), and the specificity was 99.92%(95%CI, 99.87–99.96%), 99.96%(95%CI, 99.91–99.98%), and 99.88% (95%CI, 99.82–99.93%) for the detection of trisomies 21, 18, and 13, respectively. Expanded NIPT detected 45,X, 47,XXX, 47,XXY, XYY syndrome, RATs, and CNVs with positive predictive values of 25.49%, 75%, 94.12%, 76.19%, 6.45%, and 50%, respectively. The women carrying fetuses with Trisomy 21/Trisomy 18/Trisomy 13 underwent invasive prenatal diagnosis and terminated their pregnancies at higher rates than those at high risk for SCAs, RATs, and CNVs. Conclusions Our study demonstrates that the expanded NIPT detects fetal trisomies 21, 18, and 13 with high sensitivity and specificity. The accuracy of detecting SCAs, RATs, and CNVs is still relatively poor and needs to be improved. With a high-risk expanded NIPT result, the women at high risk for common trisomies are more likely to undergo invasive prenatal diagnosis procedures and terminate their pregnancies than those with unusual chromosome abnormalities.

Download Full-text

Exome sequencing in patients with antiepileptic drug exposure and complex phenotypes

Archives of Disease in Childhood ◽

10.1136/archdischild-2018-316547 ◽

2019 ◽

Vol 105 (4) ◽

pp. 384-389 ◽

Cited By ~ 1

Author(s):

Adam Jackson ◽

Heather Ward ◽

Rebecca Louise Bromley ◽

Charulata Deshpande ◽

Pradeep Vasudevan ◽

...

Keyword(s):

Exome Sequencing ◽

Copy Number ◽

Developmental Disorders ◽

Drug Exposure ◽

Genetic Disorders ◽

Copy Number Variants ◽

Copy Number Variant ◽

In Utero ◽

Developmental Problems ◽

Number Of Children

IntroductionFetal anticonvulsant syndrome (FACS) describes the pattern of physical and developmental problems seen in those children exposed to certain antiepileptic drugs (AEDs) in utero. The diagnosis of FACS is a clinical one and so excluding alternative diagnoses such as genetic disorders is essential.MethodsWe reviewed the pathogenicity of reported variants identified on exome sequencing in the Deciphering Developmental Disorders (DDD) Study in 42 children exposed to AEDs in utero, but where a diagnosis other than FACS was suspected. In addition, we analysed chromosome microarray data from 10 patients with FACS seen in a Regional Genetics Service.ResultsSeven children (17%) from the DDD Study had a copy number variant or pathogenic variant in a developmental disorder gene which was considered to explain or partially explain their phenotype. Across the AED exposure types, variants were found in 2/15 (13%) valproate exposed cases and 3/14 (21%) carbamazepine exposed cases. No pathogenic copy number variants were identified in our local sample (n=10).ConclusionsThis study is the first of its kind to analyse the exomes of children with developmental disorders who were exposed to AEDs in utero. Though we acknowledge that the results are subject to bias, a significant number of children were identified with alternate diagnoses which had an impact on counselling and management. We suggest that consideration is given to performing whole exome sequencing as part of the diagnostic work-up for children exposed to AEDs in utero.

Download Full-text

Detection and characterization of copy number variants based on whole-genome sequencing by DNBSEQ platforms

10.1101/786962 ◽

2019 ◽

Author(s):

Junhua Rao ◽

Lihua Peng ◽

Fang Chen ◽

Hui Jiang ◽

Chunyu Geng ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Copy Number Variants ◽

Copy Number Variant ◽

Whole Genome ◽

Genome Wide ◽

Wide Range ◽

Distribution Sensitivity ◽

Cnv Detection

AbstractBackgroundNext-generation sequence (NGS) has rapidly developed in past years which makes whole-genome sequencing (WGS) becoming a more cost- and time-efficient choice in wide range of biological researches. We usually focus on some variant detection via WGS data, such as detection of single nucleotide polymorphism (SNP), insertion and deletion (Indel) and copy number variant (CNV), which playing an important role in many human diseases. However, the feasibility of CNV detection based on WGS by DNBSEQ™ platforms was unclear. We systematically analysed the genome-wide CNV detection power of DNBSEQ™ platforms and Illumina platforms on NA12878 with five commonly used tools, respectively.ResultsDNBSEQ™ platforms showed stable ability to detect slighter more CNVs on genome-wide (average 1.24-fold than Illumina platforms). Then, CNVs based on DNBSEQ™ platforms and Illumina platforms were evaluated with two public benchmarks of NA12878, respectively. DNBSEQ™ and Illumina platforms showed similar sensitivities and precisions on both two benchmarks. Further, the difference between tools for CNV detection was analyzed, and indicated the selection of tool for CNV detection could affected the CNV performance, such as count, distribution, sensitivity and precision.ConclusionThe major contribution of this paper is providing a comprehensive guide for CNV detection based on WGS by DNBSEQ™ platforms for the first time.

Download Full-text

Multiplex ligation-dependent probe amplification – a short overview

Revista Romana de Medicina de Laborator ◽

10.2478/rrlm-2020-0016 ◽

2020 ◽

Vol 28 (2) ◽

pp. 123-131

Author(s):

Valeriu Moldovan ◽

Elena Moldovan

Keyword(s):

Copy Number ◽

Genetic Disorders ◽

Cost Effective ◽

Copy Number Variations ◽

Comparative Genomic ◽

Gene Deletions ◽

Conventional Cytogenetic Analysis ◽

Main Technique ◽

Cost Effective Technique ◽

Dependent Probe

AbstractMultiplex Ligation-dependent Probe Amplification is a technique proposed for the detection of deletions or duplications that may lead to copy number variations in genomic DNA, mainly due to its higher resolution, and shorter overall diagnosis time, when compared with techniques traditionally used, namely karyotyping, fluorescence in situ hybridization, and array comparative genomic hybridization. Multiplex Ligation-dependent Probe Amplification is a fast (about 2 days), useful and cost-effective technique, being suitable for the diagnosis of hereditary conditions caused by complete or partial gene deletions or duplications, as these conditions are either more difficult or impossible to be diagnosed by other techniques, such as PCR, Real-Time PCR, or sequencing (Sanger or Next Generation). Due to its numerous advantages over conventional cytogenetic analysis techniques, Multiplex Ligation-dependent Probe Amplification could be used in the near future as the main technique for the molecular investigation of genetic conditions caused by copy number variations, in both rare and complex genetic disorders.

Download Full-text