SAM Statement of Work.docx

Statement of Work

Title: Broad Spectrum Human Papillomavirus Genotyping using Single Molecule Whole Genome Sequencing- Extended development of the bioinformatics pipeline for genotyping of human papillomavirus including calling new and known HPV variants and identification of HPV integration status

Period of performance of this contract is: 09/23/2024 – 09/22/2025.

This is a severable contract.

SECTION 1 – BACKGROUND

Human Papillomavirus (HPV) testing is essential for HPV surveillance studies of public health importance and to evaluate the burden of HPV associated cancers like cervical cancer and the impact of HPV vaccination. There are over 200 HPV genotypes, but current methods can detect only approximately 37 genotypes. There is no single laboratory test that can detect all HPV genotypes in each sample, leaving an incomplete picture in the population of the genotypes, variants, and physical status of HPV, the most sexually transmitted infectious agent. Since HPV cannot be cultured, its detection is totally dependent on molecular tools. The unmet need of developing a broad-spectrum HPV genotyping test depends essentially on advanced molecular detection (AMD) technologies and bioinformatics. On this rationale, CDC HPV laboratory at DHCPP-CVDB began developing a validated baited Single Molecule Sequencing HPV genotyping test (baited HPV-SMS genotyping test) that would allow for the detection of all known HPV genotypes, and new HPV types which is not possible with the current testing. The key steps in the development of this test will essentially involve those outlined for the human exome sequencing using Illumina HiSeq and Agilent SureSelectXT2 Target Enrichment System, but the target enrichment system consisting of biotinylated RNA library “baits” will be customized for the full-length genome (approximately 8 kb) of all known HPV types. Representing long or nearly full-length sequences of all possible HPV genomes in a sample is critical to the specificity of type-assignment, and potentially enabling the determination of variants within each genotype, critical information that is not obtained by the current genotyping methods. This new baited HPV-SMS genotyping test would subsequently be evaluated for its performance in comparison to current method(s), and compatibility with sample collection methods including formalin-fixed paraffin embedded (FFPE) samples. The HPV-SMS genotyping test will also be evaluated for performance metrics in terms of accuracy, precision, sensitivity, specificity, reproducibility, and reportable range through series of studies utilizing WHO reference materials and clinical specimens. A validated HPV-SMS genotyping test having quality performance metrics will then be piloted to HPV surveillance studies in evaluating the impact of HPV vaccination and potential for type-replacement, re-testing of HPV-negative cancers of different anatomical sites and creating a large-scale HPV whole genome sequence database using total nucleic acid (TNA) extracts from cervical specimens linked to NIH Early Detection Research Network (EDRN). The CDC-HPV laboratory has specimens from a few HPV vaccination studies and cancer registries to conduct these studies using the HPV-SMS genotyping test. The CDC HPV laboratory is currently one of two global reference laboratories, and these proposed studies using AMD technologies and bioinformatics are essential to maintaining the global reference laboratory status of the CDC-HPV lab.

CDC HPV lab, in collaboration with Agilent Technologies, completed design and production of RNA library baits (overlapping 120-mer) representing all known HPV genomes. These HPV specific RNA baits were used to capture HPV specific sequences in a pilot study using 32 samples consisting of clinical, cell lines and WHO reference materials. To capture HPV specific sequences, sample DNAs were sheared, adaptor modified, barcoded, and pooled (up to 16 samples) prior to hybridization to HPV specific RNA library baits in excess. After hybridization, background DNA was washed away, and the target HPV DNA was eluted, amplified (under reduced cycles) and pair-end sequenced by Illumina platform. In addition to this initial Illumina sequence data from 32 piloted samples, CVDB also generated HPV whole genome sequence data using (1) pooled HPV plasmids (9 HPV vaccine types) and cell lines to determine the reproducibility and limit of detection of the whole genome sequencing assay, (2) DNA extracted from external genital swabs (50 samples), (3) using hundreds of total nucleic acid extracts from EDRN biorepository completed building a largescale HPV whole genome sequence database and (4) HPV whole genome sequence data from nearly 200 formalin fixed paraffin embedded (FFPE) samples belonging to Cancer Registry for the development of bioinformatics tools for automatic determination of HPV genotype, variant and integration status in clinical specimens from epidemiologic studies. Current laboratory work is focused on optimizing the DNA library preparation and bioinformatics tools for whole genome sequencing of HPV in FFPE samples following target enrichment, and incorporation of molecular barcodes/dual indexing to address the issues of duplicates and index hoping.

SECTION 2 – PURPOSE/OBJECTIVE

CVDB has no personnel with bioinformatics expertise in the analysis of next generation sequence (NGS) data for processing of the sequence data sets from a variety of sample sources to enable HPV type and variant determination and assessment of physical status of the dominant types in each sample. The contractor from GA Tech Research Corporation participated in this project through CDC contract # 200-2017-M-95197 starting from 09/24/2016. During the contract period, the contractor has accomplished the following focusing on automating HPV variant analysis.

1. Continued in-depth analysis of the phylogenetic structure of High-risk HPVs, resulting in the development of a variant classification scheme using publicly available sequences (1376 genomes of HPV16 and 155 genomes of HPV18. Through this contract mechanism, pipeline control, interconnect between the algorithms, data download, data reformatting, quality control steps, and code for variant assignment and re-classification were implemented.

2. The trees built by pipeline are consistent with existing sub-lineage classification and is identical in topology to published trees for both HPV16 and HPV18 (PaVE).

3. For both types, HPV16 and HPV18, we detected new sub-lineages candidates. Exact location of HPV type thresholds (cut-offs) for sub-lineage classification (tree parsing) is not uniquely defined.

4. The contractor tested the tree based and minimal genome distance-based approaches for

5. classification of novel genomes into known sub-lineage groups. Both methods produced similar results. It was shown that the distance-based method was sensitive to selection of representative genome.

6. Two measures were introduced to characterize the branching node in a tree:

(within cluster distance) a mean distance between all the leaves in a branch and (between-cluster distance) a mean distance between all the leaves from two different branches. Thresholds (mean within- and between- distances, minimum number of leaves) for lineage and sub-lineage classification were determined.

7. Continued to develop the algorithm and software tool for identification of the human genome sites of HPV integration for high-risk HPV type. Three approaches are outlined to detect integration, these approaches include (1) finding reads with mixed origin, (2) finding read pairs originated from human and HPV genomes and (3) finding the integrated part of the HPV from coverage across the genome. Coverage below 100% is due to loss of HPV genome fragment during integration. Possible integration scenarios and corresponding output information include integration without loss of sequence or addition of new sequence or integration with sequence loss or addition of sequence. The accuracy of detecting the integration sites has been assessed on HeLa line, and the algorithm was extended to identify potential integration in HPV types genotyped in FFPE samples and sequenced through modified eWGS protocol and NovaSeq 6000.

The contractor tested HPV genotyping pipeline developed earlier with XT2 target enrichment protocol with NGS data generated with XT protocol and NovaSeq sequencing using FFPE DNA. Because of the substantial changes in the eWGS protocol and quality of the FFPE DNA, it was demonstrated that the existing pipeline requires modification, particularly, for processing genotyping samples with low HPV viral load.

SECTION 3 – SCOPE OF WORK

This current contract will end on 9/23/2024, and CDC DHCPP/CVDB needs to continue the bioinformatics services from this contractor for another year to expand the application of pipeline for determination of variant and integration status, in addition to genotyping HPV. The already conducted research opened new horizons in terms of additional avenues of research that would make the outcomes more precise with respect to changing technology and new volumes of accumulated data on HPV whole genome sequencing at CDC.

As part of the statement of work (SOW) during the renewal period of consultancy, the contractor will continue to provide expertise and direction to develop work-flows, and novel algorithms to automate determination of HPV types, variants and its physical status (integration of HPV genome into human genome) using whole genome sequence data from multiple sources including challenging materials like FFPE; generating multiple levels of information from the sequence data which is not dependent on the library preparation or sequencing platforms (HiSeq vs MiSeq vs Nextseq 2000). Workflows and algorithm development will take into consideration of evaluating multiple parts of the whole genome and single and multiple infections of HPV types in a clinical sample. A main thrust and outcome of this consultancy will be to continue development of the customized bioinformatics pipeline that automates determination of genotype, variant and integration status of HPV in each sample. We expect several bioinformatics challenges in developing reliable algorithms for HPV genotyping, variant assignment, and determination of integration status in a single pipeline. However, CVDB expects these challenges within the expertise of the contractor and thus their continued participation as a consultant through the GA Tech Research Corporation is highly recommended to make continued progress on this project.

Important part of the work in the next period will be to continue expanding the application of bioinformatics pipeline for determination of HPV variant and integration status and making the pipeline application independent of sequencing platforms. During the renewal period, the contractor will continue to provide the following deliverables that require high level of expert knowledge in bioinformatics: With help of this bioinformatics support service, CVDB aims to develop a molecular assay that would predict at risk HPV infected subjects towards carcinogenesis, and when to intervene to disrupthe cancer progression and save the lives independent of HPV type and anatomical site of infection.

SECTION 4 – TASKS TO BE PERFORMED

Important part of the work in the next period will be to continue expanding the application of bioinformatics pipeline for determination of HPV variant and integration status and making the pipeline application independent of sequencing platforms. During the renewal period, the contractor will continue to provide the followintasksthat require high level of expert knowledge in bioinformatics:

1. Continue working to improve the sensitivity and specificity of automated genotyping of eWGS data generated using FFPE DNA and modified library preparation and sequencing. DNA extracted from FFPE specimens are usually fragmented and low yield. FFPE DNA are known to perform poorly in molecular assays including NGS. We address this concern in our NGS library preparation protocol to improve the sensitivity of HPV detection. However, bioinformatics algorithm like machine learning is required to separate the noise from the signal for automated calling of any HPV type present at least 10-25 copies in a sample from the current sensitivity of 625 copies per sample.

2. Produce automated identification on HPV types, and HPV variant status using the algorithms and pipelines developed by the contractor for EDRN Variant analysis will include all high-risk HPV types (14 types), on all EDRN2 samples The contractor will provide to CDC EDRN2 variant data, that requires minimal additional analysis in terms of publication. This would include algorithm for identification HPV variants from incomplete sequence data. Small number of reads (just above HPV type low detection limit) could carry variant specific mutations. A filtering algorithm augmented by the alignment-based approach will identify the lineage and sub-lineage of the virus present in a sample. The algorithm will work in top-down iterations with possible types, lineages and sub-lineages determined along the steps of the process. Identify variant specific SNPs of all high-risk types from the EDRN2 data.

3. Continue work on implementing an alignment-based asoftware tool able to identify sites of integration of HPV into a human genome from analysis of the NGS short reads. .

4. Continue to assess the accuracy (of prediction of the HPV integration sites by computational modeling of NGS short reads. T The prediction should at least have 90% agreement with experimentally observed host viral integration sites. The analysis of the human genome integration sites will reveal conserved sequence patterns facilitating integration of 14 high risk HPV types

5. The contractor shall generate automated calling on integration events for high-risk HPV types from NGS data available for EDRN and Cancer Registry samples.

6. Prepare manuscripts for publication describing (a) the patterns of HPV integration into human genome revealed by the bioinformatics analysis of the NGS short reads and (b) identification of the molecular features that may contribute to the high-risk propensity for carcinogenesis of certain HPV types/variants.

7. Assemble the final pipeline to automate genotype, variant and integration of status of an individual sample using eWGS data. The final pipeline should be sensitive and specific to the molecular features of virus identified from the sequence data.

SECTION 5 – GOVERNMENT FURNISHED PROPERTY

NA

SECTION 6 – PLACE OF PERFORMANCE AND CONTRACT SUPPORT HOURS

SUBSECTION A – PLACE OF PERFORMANCE

The work can perform at the two locations:

a. Georgia Tech Resource Corporation Atlanta, GA

b. Centers for Disease Control and Prevention Atlanta, GA

SUBSECTION B – CONTRACT SUPPORT HOURS

Monday through Friday, except for federal holidays (8am-5pm EST) DELIVETABLES: Deliverables expected from the contractor are listed in the along with proposed delivery date.

the progress made toward automated variant determination (see new task 2corresponding to this deliverable

Electronic copy of written report

written report

February, 2025

s made towards determination of HPV integration status Electronic copy of written report

the progress and new direction of project.

Electronic copy of written report

SUBSECTION B – PROCUREMENT POINT OF CONTACT (POC) INFORMATION

TBD

Electronic and Information Technology Accessibility Notice

(a) Section 508 of the Rehabilitation Act of 1973 (29 U.S.C. 794d), as amended by the Workforce Investment Act of 1998 and the Architectural and Transportation Barriers Compliance Board Electronic and Information (EIT) Accessibility Standards (36 CFR part 1194), require that when Federal agencies develop, procure, maintain, or use electronic and information technology, Federal employees with disabilities have access to and use of information and data that is comparable to the access and use by Federal employees who are not individuals with disabilities, unless an undue burden would be imposed on the agency. Section 508 also requires that individuals with disabilities, who are members of the public seeking information or services from a Federal agency, have access to and use of information and data that is comparable to that provided to the public who are not individuals with disabilities, unless an undue burden would be imposed on the agency.

(b) Accordingly, any offeror responding to this solicitation must comply with established HHS EIT accessibility standards. Information about Section 508 is available at http://www.hhs.gov/web/508. The complete text of the Section 508 Final Provisions can be accessed at http://www.access-board.gov/sec508/standards.htm.

(c) The Section 508 accessibility standards applicable to this contract are: 1194.

205 WCAG 2.0 Level A & AA Success Criteria 302 Functional Performance Criteria 502 Inoperability with Assistive Technology 503 Applications 504 Authoring Tools 602 Support Documentation 603 Support Services

In order to facilitate the Government's determination whether proposed EIT supplies meet applicable Section 508 accessibility standards, offerors must submit an HHS Section 508 Product Assessment Template, in accordance with its completion instructions. The purpose of the template is to assist HHS acquisition and program officials in determining whether proposed EIT supplies conform to applicable Section 508 accessibility standards. The template allows offerors or developers to self-evaluate their supplies and documentation detail - whether they conform to a specific Section 508 accessibility standard, and any underway remediation efforts addressing conformance issues. Instructions for preparing the HHS Section 508 Evaluation Template are available under Section 508 policy on the HHS Web site http://hhs.gov/web/508.

In order to facilitate the Government's determination whether proposed EIT services meet applicable Section 508 accessibility standards, offerors must provide enough information to assist the Government in determining that the EIT services conform to Section 508 accessibility standards, including any underway remediation efforts addressing conformance issues.

(d) Respondents to this solicitation must identify any exception to Section 508 requirements. If an offeror claims its supplies or services meet applicable Section 508 accessibility standards, and it is later determined by the Government, i.e., after award of a contract or order, that supplies or services delivered do not conform to the accessibility standards, remediation of the supplies or services to the level of conformance specified in the contract will be the responsibility of the Contractor at its expense.

(e) Electronic content must be accessible to HHS acceptance criteria. Checklist for various formats is available at http://508.hhs.gov/, or from the Section 508 Coordinator listed at https://www.hhs.gov/web/section-508/additional-resources/section-508-contacts/index.html. Materials that are final items for delivery should be accompanied by the appropriate checklist, except upon approval of the Contracting Officer or Representative.