# Fragment Size-Based Pathogen Enrichment in cfDNA

> **NIH NIH R21** · UNIVERSITY OF WASHINGTON · 2021 · $233,250

## Abstract

Project Summary
Pathogen enrichment by fragment size selection of cell-free DNA
Cell-free DNA sequencing of plasma and sterile body fluids is an attractive approach for
broad-based pathogen detection. Plasma cfDNA can sample a variety of body sites and
allows for predictable and rapid (24-hour) results for agnostic pathogen detection, while
existing screening regimens such as blood culture require 5 days or more to return
negative. A major issue with shotgun sequencing approaches to infectious disease
diagnostics is the high host background induced by inflammation can significantly
compromise the analytical sensitivity of the assay. Human cfDNA forms a stereotyped
fragment size distribution with a mode of 167 bp with fewer than ~5% of fragments
measuring under 120 bp. Pathogen cfDNA is often overwhelmingly found in these
shorter fragments as they rarely protect their DNA with nucleosomes. Here, we propose
to optimize methods to selectively sequence short cfDNA fragments to increase the
analytical sensitivity of cfDNA sequencing for pathogen detection. Specifically, we will
optimize plasma extraction, library preparation, and size selection parameters to recover
shorter fragments and deplete fragments > 120bp. We will then validate this approach
on a series of 150 plasma specimens known to be positive for pathogens covering DNA
viruses, bacteria, fungi/molds as well as 100 negative plasma specimens. Using this
approach, our aim is to extend the analytical sensitivity of cfDNA sequencing to surpass
that of specific qPCR approaches.

## Key facts

- **NIH application ID:** 10199981
- **Project number:** 5R21AI153581-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Alexander L Greninger
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $233,250
- **Award type:** 5
- **Project period:** 2020-06-22 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10199981

## Citation

> US National Institutes of Health, RePORTER application 10199981, Fragment Size-Based Pathogen Enrichment in cfDNA (5R21AI153581-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10199981. Licensed CC0.

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