Preprocessing

The Preprocessing tab is the first step in the MCNV2 workflow. It performs CNV annotation and inheritance status calculation.

Overview

This tab allows you to:

Upload input files (CNV calls, pedigree, problematic regions)
Set inheritance parameters (overlap threshold, genome build)
Annotate CNVs with genes, LOEUF scores, and problematic regions
Calculate inheritance status (transmitted vs non-transmitted)
View annotated results

Input files

CNV file (mandatory)

Tab-delimited file with columns: CHR, START, STOP, TYPE (DEL/DUP), SAMPLE_ID

See Input formats for detailed specifications.

Pedigree file (mandatory)

Three-column file: SAMPLE_ID, FATHER_ID, MOTHER_ID

Only complete trios are analyzed.

Problematic regions file (optional, BED format)

BED file with problematic genomic regions:

Segmental duplications
Centromeres
Telomeres
HLA region

Note

A default file is provided. You can replace it with your own BED file if needed.

Parameters for inheritance calculation

Inheritance threshold (child CNV proportion)

Default: 0.5 (50%)

This parameter defines the minimum reciprocal overlap required between a child CNV and a parental CNV to consider the child CNV as inherited.

Value range: 0.01 to 1.0 (1% to 100%)
Interpretation: If ≥X% of a child CNV overlaps with a parental CNV (father or mother), the CNV is classified as inherited (True)
Example: With threshold=0.5, a child CNV is inherited if at least 50% of it overlaps with a CNV in at least one parent

Genome build

Default: GRCh38/hg38
Planned: GRCh37/hg19 (not yet implemented)

Workflow

Step 1: Upload files

Upload your CNV file, pedigree file, and optionally a custom problematic regions file.

Step 2: Set parameters

Inheritance threshold: Adjust the reciprocal overlap percentage (default 50%)
Genome build: Select GRCh38/hg38

Step 3: Submit

Click Submit to start annotation and inheritance calculation.

Annotation process

MCNV2 annotates each CNV with:

Gene annotation

Each CNV is intersected with gene coordinates (Gencode v45):

GeneName — HGNC gene symbol
GeneID — Ensembl gene ID
Transcript — Ensembl transcript ID

If a CNV overlaps multiple genes, one row is created per gene (CNV-gene pairs).

LOEUF scores (gnomAD v4)

LOEUF (Loss-of-function Observed/Expected Upper bound Fraction) quantifies gene constraint:

Low LOEUF (≤0.6): Highly constrained genes (intolerant to loss-of-function)
High LOEUF (>0.6): Less constrained genes

LOEUF is used for:

Stratifying Mendelian Precision by gene constraint
Optional filtering (exclude constrained genes to focus on technical precision)

Problematic regions overlap

Percentage of CNV overlapping with problematic regions:

Segmental duplications
Centromeres
Telomeres
HLA region

Note

This percentage is used in the filtering step to exclude CNVs with high overlap (e.g., >50%).

Annotated CNV table

After clicking Submit, the first table displays annotated CNVs:

Columns:

Original CNV file columns (CHR, START, STOP, TYPE, SAMPLE_ID, quality scores, etc.)
GeneName — Overlapping gene name
GeneID — Ensembl gene ID
Transcript — Ensembl transcript ID
LOEUF — Constraint score
problematic_region_overlap — Percentage overlap with problematic regions

Table navigation:

Show 10, 50, 100, or all entries
Scroll horizontally to view all columns

File path:

The file path where the annotated table is saved is displayed below the table.

Inheritance status calculation

Click Proceed to inheritance status to calculate transmission for each CNV.

MCNV2 uses two complementary approaches to determine inheritance:

Transmitted_CNV (coordinate-based) — Based on genomic coordinate overlap
Transmitted_gene (gene-based) — Based on shared affected genes

Inheritance status table

The second table displays inheritance results:

Columns:

All columns from the annotated CNV table
Transmitted_CNV — True/False (coordinate-based inheritance)
Transmitted_gene — True/False/intergenic (gene-based inheritance)

Interpretation:

True (both columns): CNV is inherited from at least one parent
False (Transmitted_CNV): Candidate de novo CNV (no parental overlap)
False (Transmitted_gene): Gene not affected in parents
“intergenic” (Transmitted_gene): CNV does not overlap any gene

File path:

The file path where the inheritance table is saved is displayed below the table.

Next step: Mendelian Precision analysis

Once inheritance status is calculated, click Go to Mendelian Precision analysis to:

Compute Mendelian Precision across size ranges and quality thresholds
Apply filters (quality scores, problematic regions, LOEUF, caller concordance)
Generate publication-ready plots

See Mendelian Precision for details on the MP analysis workflow.

Tips

Understanding the two inheritance approaches

Both Transmitted_CNV (coordinate-based) and Transmitted_gene (gene-based) have different strengths:

Transmitted_CNV requires an overlap threshold but works for all CNVs
Transmitted_gene is more robust to breakpoint and CNV fragmentation but only works for genic CNVs

Recommendation: Use both approaches to get a comprehensive view of CNV inheritance.

See Inheritance status for detailed comparison, advantages, limitations, and use cases.

Filtering intergenic CNVs

Intergenic CNVs can only be evaluated using coordinate-based matching (Transmitted_CNV).

Table too large to display

Use pagination (show 10/50/100 entries).

Troubleshooting

Error: “No complete trios found”

Check that:

Pedigree file has correct format (SAMPLE_ID, FATHER_ID, MOTHER_ID)
All three IDs (child, father, mother) are present in the pedigree file
Sample IDs are consistent between CNV and pedigree files

Warning: “Some samples in pedigree not found in CNV file”

This is normal. If a sample has no detected CNVs, it won’t appear in the CNV file. The trio is still valid.

Table too large to display

Use pagination (show 10/50/100 entries) or download the full table for offline analysis.