Submitting Next Generation 
Sequencing Data to the Division of 
Antiviral Products 
 
Guidance for Industry 
Technical Specifications Document 
 
 
 
 
 
For questions regarding this technical specifications document, contact 
CDER at cder-edata@fda.hhs.gov. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
July 2019 
Technical Specifications 
   
Revision History 
 
Date  Version  Summary of Revisions 
July 2019  1.0  Initial Version 
     
     
     
 
  Contains Nonbinding Recommendations 
Table of Contents 
1.0  Introduction .......................................................................................................................... 1 
2.0  Acceptable NGS Platforms .................................................................................................. 2 
3.0  Summary of Information to Submit to the Division ............................................................ 2 
3.1  NGS Protocol and Data Analysis Methods ...................................................................... 2 
3.2  Raw NGS Data in Fastq Format ....................................................................................... 2 
3.3  Frequency Tables ............................................................................................................. 2 
3.4  Summary Tables ............................................................................................................... 3 
3.5  Other Analysis-Ready Datasets (as applicable) ............................................................... 3 
4.0  NGS Protocol ....................................................................................................................... 3 
4.1  General Protocol Design Elements .................................................................................. 3 
4.2  Sample Preparation .......................................................................................................... 4 
5.0  Describing NGS Data Analysis Methods and Reporting Results ........................................ 4 
5.1  Summary Statistics for Each Sequence Run .................................................................... 4 
5.2  A Description of How Sequence Barcodes Were Processed ........................................... 5 
5.3  Contig and Mapping Reports ........................................................................................... 5 
5.3.1  Mapping sequence reads to a reference sequence and calling variants .................... 5 
5.3.2  Reporting results from the read mapping and variant calling ................................... 5 
5.3.3  De novo assembly of contigs and calling variants (when applicable) ...................... 6 
5.3.4  Reporting results from the de novo assembly and variant calling ............................ 6 
6.0  NGS File Types and Submission Procedures ...................................................................... 7 
6.1  Files Submitted Via Portable Hard Drive ........................................................................ 7 
6.1.1  Contents of the portable hard drive ........................................................................... 7 
6.1.2  Labeling fastq sequence files .................................................................................... 7 
6.2  Files submitted via the CDER electronic document gateway .......................................... 8 
7.0  Frequency Table Example ................................................................................................... 8 
  Contains Nonbinding Recommendations 
Submitting Next Generation Sequencing Data  
to the Division of Antiviral Products 
 
Guidance for Industry 
Technical Specifications Document1 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
1.0  Introduction 
 
The purpose of this technical specifications document is to provide the current thinking of FDAs 
Division of Antiviral Products (the Division) in regard to the submission of next generation 
nucleotide sequence analysis procedures and data in support of resistance assessments for the 
development of antiviral drug products.  
 
The Division performs independent analyses of all resistance data associated with antiviral drug 
products being developed to ensure that the emergence of resistance is carefully characterized 
and explained in the label of newly approved antiviral drug products. Providing accurate 
resistance information is imperative for protecting public health to prevent the emergence of 
novel resistant and cross-resistant viral variants that have the potential to infect others and cause 
major outbreaks of disease that cannot be controlled by approved drug products. In addition, the 
resistance information provides important guidance for health care professionals who oversee the 
use of antiviral drug products and is included in the drug product information approved by the 
Division. Moreover, the Division can request data for nucleotide sequence analysis of host-
targeted genes for polymorphism analysis to determine if different population-based alleles have 
an effect on efficacy. 
 
Next generation sequencing (NGS) is an emerging technology that sponsors frequently employ 
when performing sequence-based resistance analysis. Because this technology generates the 
nucleotide sequence for all RNAs or DNAs in a clinical sample, NGS adds complexity to the 
resistance analysis process. In contrast to Sanger nucleotide sequence analysis, which provides 
an average sequence of the virus population, NGS provides nucleotide sequence information for 
                                                 
1 This technical specifications document has been prepared by the Division of Antiviral Products in the Center for 
Drug Evaluation and Research at the Food and Drug Administration. You may submit comments on this guidance at 
any time. Submit comments to Docket No. FDA-2017-D-6821 (available at 
https://www.regulations.gov/docket?D=FDA-2017-D-6821) (see the instructions for submitting comments in the 
docket). 
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individual viruses within a viral population, frequently providing millions or billions of short 
sequences per sample. The complexity of the data makes it challenging for reviewers to analyze 
and validate the sequence information particularly because there are currently no standardized 
bioinformatics analysis approaches for analyzing these large datasets.  
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
as recommendations, unless specific regulatory or statutory requirements are cited. The use of 
the word should in Agency guidances means that something is suggested or recommended, but 
not required.  
 
2.0  Acceptable NGS Platforms 
 
The Division will accept nucleotide sequencing data generated from most standard NGS 
platforms provided 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. We recommend the sponsor communicate with the Division early in the 
process and provide these details before submitting the sequencing data. In addition, we 
recommend the sponsor submit a mock NGS dataset before any formal submissions to ensure 
that the appropriate data formats and processes are acceptable. Sponsors should consider the 
information in the following sections when preparing NGS submissions.  
 
3.0  Summary of Information to Submit to the Division 
 
3.1 NGS Protocol and Data Analysis Methods 
 
The sponsor should submit a detailed protocol that describes sample processing and NGS 
analysis procedures. See sections 4.0, NGS Protocol, and 5.0, Describing NGS Data Analysis 
Methods and Reporting Results, for specific methods to be described.   
 
3.2 Raw NGS Data in Fastq Format 
 
The sponsor should provide all of the raw NGS data from each sequence run in the fastq format. 
An assembled read mapping can be submitted in .fas, .ace, .sam, or .bam formats, but this is 
optional. The sponsor should provide the appropriate reference sequences (with concurrence 
from the division) and accession numbers used for any reference mappings. For reference 
mapping to a baseline sample or gene of interest, the sponsor should provide the baseline or 
reference consensus sequence and state how this sequence was derived. For de novo assemblies, 
the sponsor should provide all contigs greater than 200 nucleotides in the fastq format. 
 
3.3 Frequency Tables 
 
The sponsor should provide a frequency table reporting all amino acid substitutions that differ 
from baseline at frequencies greater than or equal to 1 percent. See section 7.0, Frequency Table 
Example, for an example of a frequency table.   
 
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3.4 Summary Tables 
 
The sponsor should consider the following: 
 
a.  Provide the data for each study in the analysis-ready virology resistance data format used 
for population-based sequencing showing the baseline and failure sequences and using 
blank cells when amino acids are identical to the reference sequence. We recommend that 
the sponsor consult with the Division to establish the sensitivity level for the resistance 
dataset (table populated with amino acid substitutions present at a specific frequency, i.e., 
15 percent) or provide justification for the sensitivity level selected. 
 
b.  Provide a table presenting a high-level summary of the predominant substitutions for 
each study or study subgroup (i.e., by genotype, by dose). 
 
3.5 Other Analysis-Ready Datasets (as applicable) 
 
We recommend that the sponsor consult with the Division before submitting other analysis-ready 
datasets related to the resistance analysis of NGS data. 
 
4.0  NGS Protocol 
 
4.1 General Protocol Design Elements 
 
Sponsors should include the following general protocol design elements in NGS protocols:  
 
a.  A description of the subjects, study time points, and sample matrices to be analyzed. 
 
b.  A description of the NGS platform to be used including all associated performance 
characteristics. 
 
c.  Target gene region name(s) and size(s) to be analyzed. 
 
d.  General analysis strategy (e.g., identify changes relative to a prototypical strain, compare 
sequences from different time points in the same subject). 
 
e.  The coverage level to be attempted. We recommend a target for coverage of greater than 
5,000 reads. However, we recognize that some samples with lower viral loads may not 
produce assemblies at this level of coverage. Sponsors should identify samples that did 
not reach this level of coverage.  
 
f.  A description of the approach used to identify, filter, or process sequencing errors.  
 
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4.2 Sample Preparation 
 
The key to reliable sequencing results is linked to template preparation, so that the sample being 
sequenced is representative of the population being analyzed. The following information should 
be described:  
 
a.  Methods for extracting nucleic acids from samples. 
 
b.  Methods for purifying viral sequences from contaminating background nucleic acids. 
 
c.  Methods for concentrating viral nucleic acids. Include the estimated target copy number 
input for reverse transcription polymerase chain reaction (RT-PCR) (viral RNA) or PCR 
(viral DNA) reactions for each sample. 
 
d.  Methods for denaturing secondary structure. 
 
e.  Methods for generating double stranded DNA (dsDNA). Include a description of the 
primers. 
 
f.  Methods for purifying dsDNA for sequencing. 
 
g.  Methods for NGS library preparation. 
 
h.  Methods for adding barcodes for multiplexing (when applicable). 
 
NOTE: Many NGS protocols that have been published in the scientific literature have used a 
specific (amplicon) or nonspecific (random primers) PCR amplification stage to increase the 
concentration of dsDNA for sequencing. However, while this approach will amplify the 
predominant genotypes, it is not likely to detect the minor variants that may be important for the 
rise of resistance mutations over time. Therefore, any protocol that uses a PCR amplification step 
before NGS should provide evidence that amplifications are representative of the target 
population and minor variants would still be present in the NGS data. We recommend employing 
approaches that correct for PCR resampling bias and (RT-)PCR and sequencing error, such as 
complementary DNA barcoding. 
 
5.0  Describing NGS Data Analysis Methods and 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 so that the Division can 
conduct an independent analysis of the data. The information in the following sections should be 
provided in these reports. 
 
5.1 Summary Statistics for Each Sequence Run 
 
The report should include the total number of reads sequenced per sequence run, the quality 
scores of the reads, the average length of the reads, a description of any error assessments 
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performed to reduce the effect of platform-specific errors, and information regarding what 
trimming parameters were used to clean up potential sequence errors or remove reads that failed 
to meet any quality or length thresholds. 
 
5.2 A Description of How Sequence Barcodes Were Processed 
 
The report should include a description of the methods used to add barcode sequences to the 
sequences of interest, the sequences of the barcode tags used to sequester reads after the 
sequence run, the program used to sort into bins the reads by barcode, a description of how 
barcode sequences were trimmed from the sequences, and a description of any precautions taken 
to eliminate potential cross-barcode contamination. 
 
5.3 Contig and Mapping Reports 
 
Two approaches can be used for NGS analyses of data to support the development of antiviral 
drug products: (1) mapping of short reads to a references sequence or (2) de novo assembly of 
short reads to assemble contigs. 
 
5.3.1  Mapping sequence reads to a reference sequence and calling variants 
 
Sponsors should consider the following for inclusion in the report on mapping sequence reads to 
a reference sequence and calling variants: 
 
a.  Identify the reference sequence(s) used for mapping and provide the nucleotide and 
amino acid sequences and an accession number.  
 
b.  Identify the program and describe the algorithm used to conduct the mapping of reads to 
the reference sequence and provide a list of parameter settings used along with a rationale 
for each. The report should provide specific information about the algorithm including, 
but not limited to, percentage of mismatches tolerated, number of insertions and deletions 
(indels) tolerated, and identity and similarity cutoffs.  
 
c.  Describe the algorithm employed to call variants and provide the parameters used. In 
addition, describe how single nucleotide polymorphisms (SNPs) and indels were 
distinguished from sequence errors.  
 
5.3.2  Reporting results from the read mapping and variant calling 
 
Sponsors should consider the following for inclusion in the report in results from the read 
mapping and variant calling: 
 
a.  Provide a summary of the coverage achieved for each read mapping and include a 
coverage graph.  
 
b.  Provide the number of sequences that were present in each read mapping.  
 
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c.  Provide a frequency table for all read mappings showing predominant SNPs and indels.  
 
d.  Provide a combined frequency table of the NGS results from each clinical trial in amino 
acid format that includes the following: the amino acids at each position, the frequency 
for variants at each position, and the coverage at each position where variation occurred 
(see example in section 7.0, Frequency Table Example).  
 
e.  Provide a summary table showing predominant changes across the population of interest.  
 
5.3.3  De novo assembly of contigs and calling variants (when applicable) 
 
Sponsors should consider the following for inclusion in the report on de novo assembly of 
contigs and calling variants: 
 
a.  Provide contig statistics showing the number of contigs generated from the reads.  
 
b.  Identify the program and describe the algorithm used to conduct the de novo assembly of 
short reads into contigs and provide a list of parameter settings used along with a 
rationale for each. The report should provide specific information about the algorithm 
including, but not limited to, percentage of mismatches tolerated, number of indels 
tolerated, and identity and similarity cutoffs.  
 
c.  Describe the algorithm employed to compare contigs and call variants, and provide the 
parameters used. In addition, describe how SNPs and indels were distinguished from 
sequence errors.  
 
d.  Describe how contigs were annotated by identifying the program that was used to 
identify similarity to known sequences, identify the database that was queried, provide 
the search criteria that were used, define the cutoffs used for percent identity, and provide 
details on how contaminants or other viruses were identified and reported. 
 
5.3.4  Reporting results from the de novo assembly and variant calling 
 
Sponsors should consider the following for the report on de novo assembly and variant calling: 
 
a.  Provide a summary of the coverage for each contig.  
 
b.  Provide the number of sequences in each contig and the number of reads that did not 
form contigs. 
 
c.  Describe contaminants or other viruses that were detected during annotation and report 
the percent representation of each contaminant. 
 
d.  Provide a frequency table for all major contigs showing predominant SNPs and indels.  
 
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e.  Provide a combined frequency table of the NGS results from each clinical trial in amino 
acid format that includes the following: the amino acids at each position, the frequency 
for variants at each position, and the coverage at each position where variation occurred 
(see example in section 7.0, Frequency Table Example). 
 
f.  Provide a summary table showing predominant changes across the population of interest.  
 
g.  We recommend performing de novo assembly of all reads that do not map to a reference 
sequence and annotating the contigs to assess for potential cross contamination of 
samples. 
 
6.0  NGS File Types and Submission Procedures 
 
6.1 Files Submitted Via Portable Hard Drive 
 
The raw NGS data in the fastq format and frequency tables should be sent to the Division on a 
secured, portable hard drive following the guidelines outlined in the technical specifications 
document Transmitting Electronic Submissions Using eCTD Specifications (April 2019).2 
 
6.1.1  Contents of the portable hard drive 
 
Note that only the raw NGS data, the frequency table(s), and a table of contents should be 
submitted on the hard drive. Additional files, such as those with a .exe extension, may result in 
rejection of the submission.  
 
If the hard drive is password protected (not required or recommended at this time), the sponsor 
should consult with the Division ahead of time to ensure that the password is provided to the 
appropriate personnel in the Center for Drug Evaluation and Research (CDER) document room.  
 
6.1.2  Labeling fastq sequence files 
 
All NGS data should be provided in the fastq format, which includes nucleotide sequences and 
quality information. This should include NGS sequence runs for each subject at various time 
points, including baseline and a time point close to when treatment failure was observed. The 
sponsor should provide the fastq files labeled by unique subject identifier and time point. For 
example, for study ABC123, subject 0001, and three samples (baseline, week 16, and follow up 
week 4) the files should be labeled as follows:  
 
a.  ABC123-0001.baseline.fastq  
b.  ABC123-0001.WEEK16.fastq  
c.  ABC123-0001.WEEK4FU.fastq  
 
                                                 
2 Available at https://www.fda.gov/media/76812/download. See also the Electronic Regulatory Submission and 
Review web page at 
https://www.fda.gov/Drugs/DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/d
efault.htm.  
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If a sample is resequenced, distinguish the first and second sequence runs as follows: 
 
a.  ABC123-0001.baseline-1.fastq  
b.  ABC123-0001.baseline-2.fastq  
 
6.2 Files submitted via the CDER electronic document gateway 
 
The NGS protocol, analysis-ready resistance datasets, and any other supporting information 
should be submitted via the CDER electronic document gateway. 
 
7.0  Frequency Table Example 
 
STUDYID  USUBJID  NGSPL  VISIT  AAPOS  AAREF  AASUB  TCOV  VCOV  AAFREQ 
ABC123-999  0123  Illumina  BL  81  R  K  4317  156  0.036 
ABC123-999  0123  Illumina  BL  98  K  R  2841  99  0.035 
ABC123-999  0123  Illumina  Day 2  98  K  R  9487  366  0.039 
ABC123-999  0123  Illumina  Day 3  98  K  R  9474  378  0.040 
ABC123-999  0123  Illumina  BL  120  R  Q  4310  200  0.046 
ABC123-999  0123  Illumina  Day 2  120  R  Q  12722  470  0.037 
ABC123-999  0123  Illumina  Day 3  120  R  Q  12466  489  0.039 
ABC123-999  0123  Illumina  BL  147  I  V  3456  742  0.215 
ABC123-999  0123  Illumina  Day 2  147  I  V  13456  2709  0.201 
ABC123-999  0123  Illumina  Day 3  147  I  V  13297  1934  0.145 
ABC123-999  0123  Illumina  BL  150  A  V  3107  43  0.014 
ABC123-999  0123  Illumina  Day 2  150  A  T  13116  167  0.013 
ABC123-999  0123  Illumina  BL  154  K  R  2987  124  0.042 
ABC123-999  0123  Illumina  Day 2  154  K  R  13434  1350  0.101 
ABC123-999  0123  Illumina  Day 3  154  K  R  13077  1206  0.092 
ABC123-999  0123  Illumina  Day 3  155  R  K  12459  9837  0.781 
ABC123-999  0123  Illumina  Day 3  156  P  S  13385  172  0.013 
ABC123-999  0123  Illumina  BL  186  V  I  6155  129  0.021 
ABC123-999  0123  Illumina  Day 2  186  V  I  17698  269  0.015 
ABC123-999  0123  Illumina  Day 3  186  V  I  16474  460  0.028 
ABC123-999  0123  Illumina  BL  206  K  H  9698  165  0.017 
ABC123-999  0123  Illumina  Day 2  206  K  R  24601  292  0.012 
ABC123-999  0123  Illumina  Day 3  210  S  N  23001  255  0.011 
ABC123-999  0123  Illumina  Day 3  254  H  R  25145  290  0.012 
 
STUDYID = study protocol number; USUBJID = unique subject ID; NGSPL = next generation sequencing 
platform used for sequencing; VISIT = study visit that the sample was collected from; AAPOS = amino acid 
position in the target gene; AAREF = amino acid present at this position in the reference sequence; AASUB = 
amino acid substitution detected by sequencing; TCOV = total coverage at the nucleotide site; VCOV = total 
coverage at the nucleotide position of the variant; AAFREQ = frequency of the substitution detected; BL = 
baseline. 
 
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