Analytical and Clinical Validation of the Natera In-house cfDNA Extraction (NICE) Methodology for use in Non-Invasive Prenatal Testing (Panorama™)

OBJECTIVES:

A large volume cfDNA extraction methodology (NICE) was developed and validated for use in NIPT (Panorama) performed in our CLIA-certified laboratory. Goals: No salt carryover or clogging, processing >10mL plasma. The NICE methodology was compared with the original Reference methodology used for Panorama.

METHODS:

Analysis of accuracy, precision and clinical performance was assessed for the following metrics:

• Yield Accuracy: %recovery of 72, 118, and 194bp spike fragments by qPCR
• Yield Precision: Repeatability (%CV) for each spiked fragment
• Mean filtration times
• Reduction in redraws
• Child Fraction Estimate (%CFE): Proportion of fetal DNA in maternal plasma
• Overall Error Rate (OER): Proportion of mismatched bases across all sequencing cycles
• No-call Rate: Proportion of cases where updated risk estimates were not delivered

RESULTS:

Extraction efficiency was high for both methods (see table). NICE yield ratios (%recovery NICE/%recovery Reference) were <5% reduced and slightly more variable, while mean filtration times were 9 minutes shorter. Clinical performance was assessed by analyzing 66,316 patient samples in parallel, with 43,497 samples extracted by NICE and 22,819 by Reference; all cfDNA samples were processed through the commercial Panorama pipeline. NICE yielded mean reductions in OER and no-call rate, and a mean increase %CFE of 0.59%. Increased child fraction was predicted to reduce no-call rates, findings upheld by the data and evidenced by the observed 15% reduction in redraws.

SUMMARY:

NICE was adopted for use in Natera’s CLIA laboratory and the methodology has delivered consistent performance across multiple production lots. Library and low CFE failures were reduced by 3.8- and 8.3-fold respectively, leading to 1.3% fewer redraws for the over 200,000 cases processed.