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VisCap: inference and visualization of germ-line copy-number variants from targeted clinical sequencing data.

Pugh TJ, Amr SS, Bowser MJ, Gowrisankar S, Hynes E, Mahanta LM, Rehm HL, Funke B, Lebo MS - Genet. Med. (2015)

Bottom Line: VisCap calculates the fraction of overall sequence coverage assigned to genomic intervals and computes log2 ratios of these values to the median of reference samples profiled using the same test configuration.Candidate CNVs are called when log2 ratios exceed user-defined thresholds.VisCap is a sensitive method for inferring CNVs from targeted sequence data from targeted gene panels.

View Article: PubMed Central - PubMed

Affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

ABSTRACT

Purpose: To develop and validate VisCap, a software program targeted to clinical laboratories for inference and visualization of germ-line copy-number variants (CNVs) from targeted next-generation sequencing data.

Methods: VisCap calculates the fraction of overall sequence coverage assigned to genomic intervals and computes log2 ratios of these values to the median of reference samples profiled using the same test configuration. Candidate CNVs are called when log2 ratios exceed user-defined thresholds.

Results: We optimized VisCap using 14 cases with known CNVs, followed by prospective analysis of 1,104 cases referred for diagnostic DNA sequencing. To verify calls in the prospective cohort, we used droplet digital polymerase chain reaction (PCR) to confirm 10/27 candidate CNVs and 72/72 copy-neutral genomic regions scored by VisCap. We also used a genome-wide bead array to confirm the absence of CNV calls across panels applied to 10 cases. To improve specificity, we instituted a visual scoring system that enabled experienced reviewers to differentiate true-positive from false-positive calls with minimal impact on laboratory workflow.

Conclusions: VisCap is a sensitive method for inferring CNVs from targeted sequence data from targeted gene panels. Visual scoring of data underlying CNV calls is a critical step to reduce false-positive calls for follow-up testing.Genet Med 18 7, 712-719.Genetics in Medicine (2016); 18 7, 712-719. doi:10.1038/gim.2015.156.

No MeSH data available.


Related in: MedlinePlus

Example of VisCap chromosome-level outputs. Fractional depth of coverage values for each genomic interval (black dot) sequenced on a targeted panel plotted as log2 ratios against the median of a reference set of samples analyzed using the same panel and laboratory workflow. Copy-number variants supported by multiple consecutive exons with log2 ratios outside user-defined thresholds (solid black lines at −0.55 and 0.40, in this case) are color-coded: gains are red and losses are blue. An orange marker denotes the affected genomic segment with a number corresponding to the CNV identifier listed in the output text file. This plot is overlaid with guidelines depicting the two sets of user-defined thresholds used for copy-number variation (dashed = whiskers derived from the boxplot denoting the sample's overall data distribution; solid black = fixed, user-defined thresholds) and the theoretical log2 ratios (solid light gray) for single-copy gains and single-copy losses. Labels on the x-axis mark the first interval of each group of exons (commonly a gene name) as specified in the list of intervals provided to the program (e.g., TP53_Exon1 and TP53_Exon2 would be marked by a single TP53 label underneath exon 1). Panels a–d contain examples of VisCap output plots from four cases selected from Table 1. (a) Whole-genome view depicting log2 ratios from all intervals from a single patient run on a large gene panel. This individual had a GJB6 deletion known from previous testing as well as an unexpected gain of chromosome X. The patient was phenotypically female and had median log2 ratio of X-chromosome probes twice that of other females, suggesting a potentially undiagnosed sex chromosome abnormality. (b) Single-chromosome view of data indicating a full gene, single-copy deletion of OTOF in a patient with a loss-of-function mutation on the remaining allele, illustrating compound heterozygosity resulting in nonsyndromic hearing loss. (c) Example of a multi-exon copy-number gain within FBN1 leading to Marfan syndrome. (d) Example of two homozygous, noncontiguous exon-level deletion calls within TMPRSS3 in a patient with nonsyndromic hearing loss. Family testing found that both of the patient's parents were heterozygous for both deletion calls, suggesting the deletions are on the same allele and may be indicative of a complex rearrangement.
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fig1: Example of VisCap chromosome-level outputs. Fractional depth of coverage values for each genomic interval (black dot) sequenced on a targeted panel plotted as log2 ratios against the median of a reference set of samples analyzed using the same panel and laboratory workflow. Copy-number variants supported by multiple consecutive exons with log2 ratios outside user-defined thresholds (solid black lines at −0.55 and 0.40, in this case) are color-coded: gains are red and losses are blue. An orange marker denotes the affected genomic segment with a number corresponding to the CNV identifier listed in the output text file. This plot is overlaid with guidelines depicting the two sets of user-defined thresholds used for copy-number variation (dashed = whiskers derived from the boxplot denoting the sample's overall data distribution; solid black = fixed, user-defined thresholds) and the theoretical log2 ratios (solid light gray) for single-copy gains and single-copy losses. Labels on the x-axis mark the first interval of each group of exons (commonly a gene name) as specified in the list of intervals provided to the program (e.g., TP53_Exon1 and TP53_Exon2 would be marked by a single TP53 label underneath exon 1). Panels a–d contain examples of VisCap output plots from four cases selected from Table 1. (a) Whole-genome view depicting log2 ratios from all intervals from a single patient run on a large gene panel. This individual had a GJB6 deletion known from previous testing as well as an unexpected gain of chromosome X. The patient was phenotypically female and had median log2 ratio of X-chromosome probes twice that of other females, suggesting a potentially undiagnosed sex chromosome abnormality. (b) Single-chromosome view of data indicating a full gene, single-copy deletion of OTOF in a patient with a loss-of-function mutation on the remaining allele, illustrating compound heterozygosity resulting in nonsyndromic hearing loss. (c) Example of a multi-exon copy-number gain within FBN1 leading to Marfan syndrome. (d) Example of two homozygous, noncontiguous exon-level deletion calls within TMPRSS3 in a patient with nonsyndromic hearing loss. Family testing found that both of the patient's parents were heterozygous for both deletion calls, suggesting the deletions are on the same allele and may be indicative of a complex rearrangement.

Mentions: The initial step in the VisCap program is to generate a matrix of all intervals captured and the fraction of total coverage assigned to these intervals. These are derived for each sample from DepthOfCoverage “sample_interval_summary” files using total coverage values in the column “{sample name}_total_cvg”. Next, the sample-specific fractional coverage of each region is divided by the median for that region across the entire batch, where the number of samples to be batched together can be any size that provides a representative median coverage across all target regions. In our clinical workflow, a batch refers to a set of 7–10 DNA samples captured in a single multiplexed pool and sequenced in a single flow-cell lane of an Illumina HiSeq 2000 or 2500 DNA sequencer. These batch median-normalized data are stored as a matrix of log2 ratios that is written to a text file. To facilitate visual review of the data for each sample, log2 ratios are plotted by relative genome order (i.e., rank order, not to scale on genome), and data points supporting CNVs are color-coded and linked to a unique identifier listed in the text output (Figure 1).


VisCap: inference and visualization of germ-line copy-number variants from targeted clinical sequencing data.

Pugh TJ, Amr SS, Bowser MJ, Gowrisankar S, Hynes E, Mahanta LM, Rehm HL, Funke B, Lebo MS - Genet. Med. (2015)

Example of VisCap chromosome-level outputs. Fractional depth of coverage values for each genomic interval (black dot) sequenced on a targeted panel plotted as log2 ratios against the median of a reference set of samples analyzed using the same panel and laboratory workflow. Copy-number variants supported by multiple consecutive exons with log2 ratios outside user-defined thresholds (solid black lines at −0.55 and 0.40, in this case) are color-coded: gains are red and losses are blue. An orange marker denotes the affected genomic segment with a number corresponding to the CNV identifier listed in the output text file. This plot is overlaid with guidelines depicting the two sets of user-defined thresholds used for copy-number variation (dashed = whiskers derived from the boxplot denoting the sample's overall data distribution; solid black = fixed, user-defined thresholds) and the theoretical log2 ratios (solid light gray) for single-copy gains and single-copy losses. Labels on the x-axis mark the first interval of each group of exons (commonly a gene name) as specified in the list of intervals provided to the program (e.g., TP53_Exon1 and TP53_Exon2 would be marked by a single TP53 label underneath exon 1). Panels a–d contain examples of VisCap output plots from four cases selected from Table 1. (a) Whole-genome view depicting log2 ratios from all intervals from a single patient run on a large gene panel. This individual had a GJB6 deletion known from previous testing as well as an unexpected gain of chromosome X. The patient was phenotypically female and had median log2 ratio of X-chromosome probes twice that of other females, suggesting a potentially undiagnosed sex chromosome abnormality. (b) Single-chromosome view of data indicating a full gene, single-copy deletion of OTOF in a patient with a loss-of-function mutation on the remaining allele, illustrating compound heterozygosity resulting in nonsyndromic hearing loss. (c) Example of a multi-exon copy-number gain within FBN1 leading to Marfan syndrome. (d) Example of two homozygous, noncontiguous exon-level deletion calls within TMPRSS3 in a patient with nonsyndromic hearing loss. Family testing found that both of the patient's parents were heterozygous for both deletion calls, suggesting the deletions are on the same allele and may be indicative of a complex rearrangement.
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Related In: Results  -  Collection

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fig1: Example of VisCap chromosome-level outputs. Fractional depth of coverage values for each genomic interval (black dot) sequenced on a targeted panel plotted as log2 ratios against the median of a reference set of samples analyzed using the same panel and laboratory workflow. Copy-number variants supported by multiple consecutive exons with log2 ratios outside user-defined thresholds (solid black lines at −0.55 and 0.40, in this case) are color-coded: gains are red and losses are blue. An orange marker denotes the affected genomic segment with a number corresponding to the CNV identifier listed in the output text file. This plot is overlaid with guidelines depicting the two sets of user-defined thresholds used for copy-number variation (dashed = whiskers derived from the boxplot denoting the sample's overall data distribution; solid black = fixed, user-defined thresholds) and the theoretical log2 ratios (solid light gray) for single-copy gains and single-copy losses. Labels on the x-axis mark the first interval of each group of exons (commonly a gene name) as specified in the list of intervals provided to the program (e.g., TP53_Exon1 and TP53_Exon2 would be marked by a single TP53 label underneath exon 1). Panels a–d contain examples of VisCap output plots from four cases selected from Table 1. (a) Whole-genome view depicting log2 ratios from all intervals from a single patient run on a large gene panel. This individual had a GJB6 deletion known from previous testing as well as an unexpected gain of chromosome X. The patient was phenotypically female and had median log2 ratio of X-chromosome probes twice that of other females, suggesting a potentially undiagnosed sex chromosome abnormality. (b) Single-chromosome view of data indicating a full gene, single-copy deletion of OTOF in a patient with a loss-of-function mutation on the remaining allele, illustrating compound heterozygosity resulting in nonsyndromic hearing loss. (c) Example of a multi-exon copy-number gain within FBN1 leading to Marfan syndrome. (d) Example of two homozygous, noncontiguous exon-level deletion calls within TMPRSS3 in a patient with nonsyndromic hearing loss. Family testing found that both of the patient's parents were heterozygous for both deletion calls, suggesting the deletions are on the same allele and may be indicative of a complex rearrangement.
Mentions: The initial step in the VisCap program is to generate a matrix of all intervals captured and the fraction of total coverage assigned to these intervals. These are derived for each sample from DepthOfCoverage “sample_interval_summary” files using total coverage values in the column “{sample name}_total_cvg”. Next, the sample-specific fractional coverage of each region is divided by the median for that region across the entire batch, where the number of samples to be batched together can be any size that provides a representative median coverage across all target regions. In our clinical workflow, a batch refers to a set of 7–10 DNA samples captured in a single multiplexed pool and sequenced in a single flow-cell lane of an Illumina HiSeq 2000 or 2500 DNA sequencer. These batch median-normalized data are stored as a matrix of log2 ratios that is written to a text file. To facilitate visual review of the data for each sample, log2 ratios are plotted by relative genome order (i.e., rank order, not to scale on genome), and data points supporting CNVs are color-coded and linked to a unique identifier listed in the text output (Figure 1).

Bottom Line: VisCap calculates the fraction of overall sequence coverage assigned to genomic intervals and computes log2 ratios of these values to the median of reference samples profiled using the same test configuration.Candidate CNVs are called when log2 ratios exceed user-defined thresholds.VisCap is a sensitive method for inferring CNVs from targeted sequence data from targeted gene panels.

View Article: PubMed Central - PubMed

Affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

ABSTRACT

Purpose: To develop and validate VisCap, a software program targeted to clinical laboratories for inference and visualization of germ-line copy-number variants (CNVs) from targeted next-generation sequencing data.

Methods: VisCap calculates the fraction of overall sequence coverage assigned to genomic intervals and computes log2 ratios of these values to the median of reference samples profiled using the same test configuration. Candidate CNVs are called when log2 ratios exceed user-defined thresholds.

Results: We optimized VisCap using 14 cases with known CNVs, followed by prospective analysis of 1,104 cases referred for diagnostic DNA sequencing. To verify calls in the prospective cohort, we used droplet digital polymerase chain reaction (PCR) to confirm 10/27 candidate CNVs and 72/72 copy-neutral genomic regions scored by VisCap. We also used a genome-wide bead array to confirm the absence of CNV calls across panels applied to 10 cases. To improve specificity, we instituted a visual scoring system that enabled experienced reviewers to differentiate true-positive from false-positive calls with minimal impact on laboratory workflow.

Conclusions: VisCap is a sensitive method for inferring CNVs from targeted sequence data from targeted gene panels. Visual scoring of data underlying CNV calls is a critical step to reduce false-positive calls for follow-up testing.Genet Med 18 7, 712-719.Genetics in Medicine (2016); 18 7, 712-719. doi:10.1038/gim.2015.156.

No MeSH data available.


Related in: MedlinePlus