Emerging Tests: FDA Issues New Guidance on Using NGS Data to Secure Antiviral Drug & CDx Test Approval

Next generation sequencing (NGS) data supporting resistance assessments is crucial to developing and gaining FDA clearance for new antiviral drugs and related companion diagnostic tests (CDx). Now the agency has issued a new Technical Specifications document (Tech Doc) telling sponsors they should use that data to support their products.

What the Tech Doc Is All About

Unlike conventional Sanger nucleotide sequence which measures viral resistance by providing an average sequence of the virus population, NGS provides nucleotide sequence information for individual viruses within a viral population, often generating millions or billions of short sequences per sample. The complexity of the data makes it hard for reviewers from the FDA’s Division of Antiviral Products to analyze and validate the sequence information. Adding to the challenge is the current lack of a standardized bioinformatics analysis approaches for analyzing such large datasets.

The purpose of the Tech Doc is to explain to sponsors how to compile, analyze and submit NGS resistance assessment data so the Division can review it. The Tech Doc provides crucial guidance on six key issues.

1. Acceptable NGS Platforms

The Tech Doc indicates that the Division will accept nucleotide sequencing data generated from most standard NGS platforms as long as the sponsor submits:

  • The appropriate details for the sequencing platform;
  • The protocols used for sample preparation;
  • The raw NGS data in fastq format; and
  • The methods used to analyze the data.

In addition to providing these details to the Division before submitting the sequencing data, sponsors should and submit a mock NGS dataset before any formal submissions to ensure

the appropriate data formats and processes are acceptable.

2. Information about NGS Protocol

The Tech Doc calls on sponsors to submit a detailed NGS protocol that includes six design elements:

  • A description of the subjects, study time points and sample matrices to be analyzed;
  • A description of the NGS platform and all associated performance characteristics;
  • Target gene region name(s) and size(s) to be analyzed;
  • A description of the general analysis strategy;
  • The coverage level to be attempted; and
  • A description of the approach used to identify, filter or process sequencing errors.

3. Frequency Tables

Sponsors should provide a frequency table reporting all amino acid substitutions that differ from baseline at frequencies greater than or equal to 1%. The Tech Doc includes the following example:

Frequency Table Example
Frequency Table Example Image

4. Sample Preparation Information

The Tech Doc instructs sponsors to list their methods for:

  • Extracting nucleic acids from samples;
  • Purifying viral sequences from contaminating background nucleic acids;
  • Concentrating viral nucleic acids, including the estimated target copy number input for reverse transcription polymerase chain reaction (RT-PCR) (viral RNA) or PCR (viral DNA) reactions for each sample;
  • Denaturing secondary structure;
  • Generating double stranded DNA (dsDNA), including a description of the primers;
  • Purifying dsDNA for sequencing;
  • NGS library preparation; and
  • Adding barcodes for multiplexing (if applicable).

5. Information about Data Analysis & Reporting Results

Submissions of sequence data must include a thorough description of the analysis pipeline used to analyze the sequencing dataset and the raw sequence information, including:

  • Summary statistics for each sequence run, including total number of reads sequenced per sequence run, quality scores and average length of reads;
  • A description of how sequence barcodes were processed;
  • Contig and mapping reports—the Tech Doc recommends two data analysis approaches and establishes standards for each: i. mapping of short reads to a reference sequence; or ii. de novo assembly of short reads to assemble contigs.

6. Acceptable Data File Types

The Tech Doc calls on sponsors to provide all raw NGS data from each sequence run in the fastq format. Sponsors can submit an assembled read mapping in .fas, .ace, .sam, or .bam formats, but this is optional.

Takeaway:Although the Tech Doc’s recommendations are not technically binding, failing to adhere to them may result in the delay or rejection of the submission. So, antiviral drug and CDx makers need to pay close attention to them.


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