Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples

Abstract

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments &lt;100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing

“…Ultrashort PCR using a stem-loop primer may be an attractive strategy for amplification of fragments too short for conventional PCR (Shekhtman et al, 2009). Alternatively, recent work by our group demonstrated that sequence-specific purification improves recovery of short cfDNA relative to conventional silica-based extraction and increases the clinical sensitivity of TB diagnosis from urine cfDNA Oreskovic and Lutz, 2021). Moreover, our results, in concert with those of a previous study (Sinkov et al, 2019), suggest that targeting multicopy genomic elements (e.g., IS6110, IS1081) is likely a more promising strategy than identification of highly represented cfDNA targets de novo.…”

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA

“…The recovery of the positive control was calculated as a percentage of the input (mean ± standard deviation [SD], n = 15). Key design features, assay optimization, and additional analytical characterization of our sequence-specific purification method for cfDNA are reported in reference 24 .…”

“…Transrenal urine cell-free DNA (cfDNA) was extracted in triplicate from 10-ml urine samples (3 × 10-ml samples for each participant), with individual replicates for each participant processed on separate days. TB-specific urine cfDNA was extracted using our in-house sequence-specific hybridization capture method, as described previously ( 24 ). We have published a detailed, user-ready protocol at http://dx.doi.org/10.17504/protocols.io.bep4jdqw .…”

“…We developed a highly sensitive sequence-specific purification method that uses DNA probes immobilized on magnetic beads to extract TB-specific cfDNA via hybridization. By combining sequence-specific purification with short-target PCR, we can reliably detect ≤5 copies of short (50 bp) cfDNA in 10 ml urine ( 24 ).…”

Unbiased sequencing of Mycobacterium tuberculosis urinary cell-free DNA reveals extremely short fragment lengths

“…To improve the clinical sensitivity of TB urine cfDNA assays, both sample preparation and amplification methods having maximal efficiency for very short fragments are needed. For example, recent work in our lab demonstrated that sequence-specific purification improves recovery of short cfDNA relative to conventional silica-based extraction and increases the clinical sensitivity of TB diagnosis from urine cfDNA (8,35). Ultrashort PCR using a stem-loop primer may be an attractive strategy for amplification of fragments too short for conventional PCR (15).…”