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The Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay is a targeted sequencing in vitro diagnostic system that re-sequences the protein coding regions and intrones of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in genomic DNA isolated from human periphers collected in K2EDTA. The test detects single nucleotide variants, and small InDels within the region sequenced, and additionally reports on two deep intronic mutations and two large deletions. The test is intended to be used on the Illumina MiSeqDx Instrument.

The test is intended to be used as an aid in the diagnosis of individuals with suspected cystic fibrosis (CF). The test is most appropriate when the patient has an atypical or non-classic presentation of CF or when other mutation panels have failed to identify both causative mutations. The results of the test are intented by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available information including clinical symptoms, other diagnostic tess, and family history. This test is not indicated for use for stand-alone diagnostic testing, for pre-implantation testing, carrier screening, newborn screening, or population screening.

The Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay consists of library preparation and sample indexing reagents, sequencing reagents and consumables, MiSeqDx instrument and data analysis software. Testing begins with genomic DNA from a peripheral whole blood sample. The genomic DNA is processed through the library preparation steps, which specifically amplifies the intended genomic regions of each sample while also adding the indexes for sample identification. Flow cell capture sequences are also added to the amplified products. The resulting sample libraries are then transferred into a MiSeqDx reagent cartridge which contains all of the reagents required for cluster generation and sequencing (Sequencing By Synthesis). The MiSeqDx Cartridge, MiSeqDx Flow Cell, and MiSeqDx SBS Solution (PR2) are then inserted into the MiSeqDx instrument, which performs cluster generation, sequencing and data analysis.

Here's a summary of the acceptance criteria and study details for the Illumina MiSeqDx™ Cystic Fibrosis Clinical Sequencing Assay based on the provided text:

Acceptance Criteria and Device Performance

Study Details

2. Sample Size Used for the Test Set and Data Provenance

• Accuracy Study Test Set:

  • Total Samples: 500
  • Clinical Samples: 355 (archived, anonymized gDNA from human blood) - Country of origin not specified, but the review is by the US FDA.
  • Commercial Cell Line Samples: 11 (from clinical accuracy study) + 68 (from reproducibility study) = 79
  • Clinical Samples (from extraction method evaluation): 14
  • Synthetic Plasmid Samples: 52
  • Data Provenance: Retrospective for archived clinical samples, prospective for cell line and synthetic samples generated for the study.

• Reproducibility Study Test Set:

  • Total Samples: 46 distinct samples (implied from the HGVS tables, with many tested in multiple replicates). The tables show analysis for 68 samples, each tested across 3 sites, with 2 operators each doing 3 runs per site, and with some samples having multiple replicates within those runs. This results in a large number of individual tests. The text explicitly states 68 cell line samples were evaluated in the reproducibility study for accuracy data.
  • Data Provenance: Prospective (generated specifically for the study). The operator details suggest this was done as part of the validation study.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

• The ground truth reference methods were Sanger bi-directional sequencing and validated PCR based assays.
• The text does not specify the number of experts used to establish the ground truth for Sanger sequencing or the PCR assays, nor their specific qualifications. It only states these were "reference methods."

4. Adjudication Method for the Test Set

• The text mentions that "All results are based on initial testing. No repeat testing was done for this study" for the accuracy study. For reproducibility, some samples had "no calls" or "miscalls" initially, and further investigation was done (e.g., misidentified variant, low level contamination, improperly prepared synthetic specimen, switched samples). However, a formal adjudication method (e.g., 2+1, 3+1) involving a specific number of experts to resolve discrepancies against the reference is not explicitly described. The determination of miscalls and no calls seems to stem from comparison with the reference methods.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

• No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done. This study focuses on the standalone analytical performance (accuracy, reproducibility) of the device (an in vitro diagnostic system for genetic sequencing), not on its impact on human reader performance.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

• Yes, a standalone performance study was done. The entire accuracy and reproducibility analysis describes the performance of the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay (which includes the instrument and data analysis software) in detecting genetic variants. The results (PA, NA, OA, call rates) are indicative of the algorithm's performance compared to established reference methods (Sanger sequencing and PCR assays). The "human-in-the-loop" aspect for interpretation is mentioned in the intended use, where results are to be interpreted by a board-certified clinical molecular geneticist, but the performance metrics provided are for the device's ability to 'call' the variants.

7. The Type of Ground Truth Used

• Expert Consensus (implied via Gold Standard Methods): The primary ground truth for SNVs and small InDels was Sanger bi-directional sequencing. For large deletions, two validated PCR-based assays were used, with accuracy confirmed by Sanger Sequencing. These are considered gold-standard methods in molecular diagnostics, implying an expert-established and validated ground truth.

8. The Sample Size for the Training Set

• The document does not specify a training set size. As this is a performance validation document for a sequencing assay, it describes the testing of the device rather than the development or training of an AI model in the conventional sense. The "MiSeqDx instrument and data analysis software" are components, but details on their development/training are not provided in this regulatory summary.

9. How the Ground Truth for the Training Set Was Established

• Since a training set is not explicitly mentioned or detailed for the device's software/algorithm development, the method for establishing its ground truth is not provided in this document. The focus is on validating the final product's performance against reference standards.

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