NIPTviewer Technical Manual

3.1 QC Metrics Summary Card

Purpose: Provides an at-a-glance summary of key quality metrics for the selected sample, enabling rapid identification of samples with compromised data quality.

Metrics Displayed:

• QC Status: Overall pass/fail determination based on laboratory-defined criteria
• GC Bias: Systematic bias related to GC content of sequenced fragments (optimal: 0.95-1.05)
• Total Reads: Number of uniquely mapped sequencing tags
• Non-Excluded Sites Ratio: Proportion of genome included in analysis after filtering

3.2 Fetal Fraction Over Time

Purpose: Tracks fetal fraction values across samples in the batch, enabling identification of samples with potentially insufficient FF for reliable screening.

Clinical Significance:

• FF < 4%: Insufficient for reliable screening; consider redraw at later gestational age
• FF 4-10%: Acceptable but may have reduced sensitivity for some conditions
• FF > 10%: Optimal sensitivity for aneuploidy detection

Visualization: Line chart showing FF values for each sample, with reference lines at 4% (minimum threshold) and 10% (optimal threshold).

3.3 Reads Distribution

Purpose: Visualizes the sequencing read processing pipeline, showing the progression from raw clusters to filtered, analysis-ready reads.

Interpretation: A healthy sample shows progressive reduction through filtering stages while maintaining adequate final read counts. Excessive loss at any stage may indicate sample or library preparation issues.

3.4 Chromosome Coverage Distribution

Purpose: Displays the sequencing coverage depth for each chromosome, enabling assessment of coverage uniformity across the genome.

Expected Pattern: Coverage values correlate with chromosome size (Chr1 highest, Chr21/22 lowest). The pattern should be consistent across samples within a batch. Deviations may indicate library bias or true biological variation.

Visual Encoding: Target chromosomes (13, 18, 21, X, Y) are highlighted in teal for easy identification.

4.1 Chromosome Z-Score Bar Chart

Purpose: Displays the Normalized Chromosome Values (Z-scores) for the five clinically relevant chromosomes, enabling rapid identification of potential aneuploidies.

View Modes:

• Single Sample: Detailed view of one sample with clinical interpretation
• All Samples: Comparison across all test samples in the batch, with batch-level warnings for any abnormal samples

Color Coding:

Clinical Associations:

• Elevated NCV_13: Trisomy 13 (Patau syndrome)
• Elevated NCV_18: Trisomy 18 (Edwards syndrome)
• Elevated NCV_21: Trisomy 21 (Down syndrome)
• Abnormal NCV_X/NCV_Y: Sex chromosome aneuploidies

4.2 Normalized Coverage Ratios

Purpose: Displays the normalized chromosome representation ratios for target chromosomes. Unlike Z-scores which are standardized to a reference population, ratios show the direct proportional representation.

Interpretation:

Example: A Ratio_21 of 1.0582 indicates ~5.8% over-representation of chromosome 21, consistent with a fetal trisomy 21 at the observed fetal fraction.

4.3 Genome-Wide CNV Profile

Purpose: Visualizes copy number variations using log₂ ratio transformation, a standard representation in genomic analysis. This approach is inspired by WisecondorX visualization methodology.

Color Encoding: Bar colors reflect NCV significance levels, providing integrated visualization of both ratio magnitude and statistical confidence.

4.4 Aberration Highlight Plot

Purpose: Provides a focused view of chromosomal aberrations with configurable Z-score thresholds and clear gain/loss classification.

Features:

• Adjustable Z-score threshold slider for customizing aberration calling sensitivity
• Color-coded bars distinguishing gains (red/orange) from losses (blue)
• Reference lines at ±2, ±3, and ±4 Z-score thresholds
• Summary badges showing total detected gains and losses

4.5 NCD Over Time

Purpose: Tracks Normalized Chromosome Deviation values across samples in the batch, showing temporal consistency and enabling identification of batch effects.

Clinical Use: Useful for monitoring run-to-run consistency and identifying systematic shifts that may indicate assay drift or batch-specific issues.

4.6 Sex Chromosome Analysis

Purpose: Scatter plot visualization of sex chromosome NCV values for fetal sex determination and sex chromosome aneuploidy detection.

Expected Patterns:

• Female fetus (46,XX): NCV_X near 0, NCV_Y negative
• Male fetus (46,XY): NCV_X slightly negative, NCV_Y elevated (correlates with FF)
• Sex chromosome aneuploidies: Deviations from expected clusters may indicate conditions such as Turner syndrome (45,X), Klinefelter syndrome (47,XXY), or others

4.7 Y-Fraction Histogram

Purpose: Histogram with Gaussian mixture model overlay for fetal sex determination, inspired by WisecondorX methodology.

Interpretation:

• Y-fraction < 0.1%: Predicted female fetus
• Y-fraction ≥ 0.1%: Predicted male fetus

The bimodal distribution with overlaid Gaussian fits allows visualization of the separation between female and male samples, with the valley representing the classification threshold.

5.1 FF vs NCV Correlation Scatter

Purpose: Visualizes the relationship between fetal fraction and NCV to assess result confidence and identify potential false positives/negatives.

Confidence Zone Assessment:

Clinical Rationale: For a true fetal trisomy, the expected NCV magnitude is proportional to fetal fraction. Low FF with high NCV may indicate confined placental mosaicism, while high FF with borderline NCV warrants careful interpretation.

5.2 Confidence Score Panel

Purpose: Provides a composite confidence score (0-100) integrating multiple quality metrics, presented as a traffic-light gauge.

Component Metrics and Weights:

• Fetal Fraction (30%): Assesses adequacy of fetal DNA content
• NCV Quality (30%): Evaluates significance of detected signals
• Sequencing Quality (25%): Incorporates coverage depth and QC flags
• Profile Uniformity (15%): Uses sex chromosome NCVs as proxy for overall profile quality

Score Interpretation:

• 80-100: High confidence - results reliable
• 50-79: Review recommended - examine individual metrics
• < 50: Low confidence - consider repeat testing

5.3 Maternal-Fetal Signal Estimator

Purpose: Assists in distinguishing whether an elevated NCV signal originates from the fetus, mother, or represents a technical artifact.

Expected NCV Estimation:

For a true fetal trisomy, the expected NCV is proportional to fetal fraction. The algorithm compares observed NCV to estimated expected values:

Signal Origin Classification:

• Fetal: Observed NCV within expected range for FF
• Maternal: Observed NCV higher than expected (suggests maternal CNV)
• Artifact: Inconsistent pattern across chromosomes

5.4 Confined Placental Mosaicism (CPM) Indicator

Purpose: Assesses the likelihood of confined placental mosaicism as an explanation for positive NIPT results.

Background: CPM occurs when chromosomal abnormality is present in the placenta but not in the fetus. Since NIPT analyzes placental cfDNA, CPM is a recognized cause of false-positive results.

CPM Risk by Chromosome:

NCV Zone Assessment:

• Gray Zone (NCV 3-5): Higher CPM probability; CVS may detect placental mosaicism
• Strong Positive (NCV >7): More likely true fetal aneuploidy; amniocentesis recommended