A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

Koker, Andries De; Paemel, Ruben Van; Wilde, Bram De; Preter, Katleen De; Callewaert, Nico

doi:10.1101/663195

Cited by 19 publications

(24 citation statements)

References 21 publications

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“…The accuracy of CelFiE depends on a number of factors including the read depth, the number and cell type-informativeness of sites considered, the abundance of key cell types, and the quantity and quality of reference data and cfDNA patient samples. Recent technologies for digesting or capturing specific regions of cfDNA [50], may allow deeper sequencing at informative CpGs. Our algorithm for selecting such TIM CpGs demonstrated marked improvement in accuracy and could be used to select sites for capture.…”

Section: Discussionmentioning

confidence: 99%

Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

Caggiano

Celona

Garton

et al. 2020

Preprint

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Circulating cell-free DNA (cfDNA) in the bloodstream originates from dying cells and is a promising non-invasive biomarker for cell death. Here, we develop a method to accurately estimate the relative abundances of cell types contributing to cfDNA. We leverage the distinct DNA methylation profile of each cell type throughout the body. Decomposing the cfDNA mixture is difficult, as fragments from relevant cell types may only be present in a small amount. We propose an algorithm, CelFiE, that estimates cell type proportion from both whole genome cfDNA input and reference data. CelFiE accommodates low coverage data, does not rely on CpG site curation, and estimates contributions from multiple unknown cell types that are not available in reference data. In simulations we show that CelFiE can accurately estimate known and unknown cell type of origin of cfDNA mixtures in low coverage and noisy data. Simulations also demonstrate that we can effectively estimate cfDNA originating from rare cell types composing less than 0.01% of the total cfDNA. To validate CelFiE, we use a positive control: cfDNA extracted from pregnant and non-pregnant women. CelFiE estimates a large placenta component specifically in pregnant women (p = 9.1 × 10 −5 ). Finally, we use CelFiE to decompose cfDNA from ALS patients and age matched controls. We find increased cfDNA concentrations in ALS patients (p = 3.0 × 10 −3 ). Specifically, CelFiE estimates increased skeletal muscle component in the cfDNA of ALS patients (p = 2.6 × 10 −3 ), which is consistent with muscle impairment characterizing ALS. Quantification of skeletal muscle death in ALS is novel, and overall suggests that CelFiE may be a useful tool for biomarker discovery and monitoring of disease progression.

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Section: Discussionmentioning

confidence: 99%

Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

Caggiano

Celona

Garton

et al. 2020

Preprint

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“…However, this technique has a lower cost-effectiveness compared to cf-RRBS, is difficult to implement in a routine clinical setting and the protocol is cumbersome and timeconsuming 17 . Single cell RRBS (scRRBS) has also been suggested as a cost-effective alternative to classical RRBS and WGBS for low-input samples, but is more laborintensive 15 .…”

Section: Discussionmentioning

confidence: 99%

“…Enrichment of CpG-rich sites by digesting DNA with the MspI restriction enzyme (cuts 5'-C/CGG-3'), followed by DNA fragment size-selection is a commonly applied strategy of RRBS. This method was redesigned by De Koker et al 17 to allow single tube RRBS library preparation on low input levels (10 ng) of low-quality and/or fragmented DNA.…”

Section: Introductionmentioning

confidence: 99%

Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

Paemel

Koker

Vandeputte

et al. 2019

Preprint

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In the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA.Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

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“…Library construction was performed according to the methods described by De Koker et al 17 with the following 3 modifications: [1] The complete eluate that was remaining after quality control (2 μL Qubit and 2 μL Femto PULSE) was used for library construction (= 71 μL). For this, samples were concentrated with a vacuum centrifuge (SpeedVac, Thermo Fischer Scientific, V-AQ program) at 35 °C and nuclease-free water was added to a volume of 11.1 μL.…”

Section: Methodsmentioning

confidence: 99%

“…To our knowledge, no study has systematically evaluated the effect of various preservation agents and the timing of plasma preparation after blood draw on the genome-wide methylation pattern. To this end, we have collected 45 blood samples in 5 different blood collection tubes (4 preservation tubes and 1 EDTA tube) from 3 healthy volunteers and performed plasma preparation at 3 different time points after blood draw followed by reduced representation bisulfite sequencing on the resulting cell-free DNA (cf-RRBS) 17 .…”

Section: Introductionmentioning

confidence: 99%

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

Paemel

Koker

Caggiano

et al. 2020

Preprint

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BackgroundThe methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulfite-based cfDNA methylation profiling.Methods15 tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulfite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics.ResultsAll preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes.ConclusionThe methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

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A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

Cited by 19 publications

References 21 publications

Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

Estimating the rate of cell type degeneration from epigenetic sequencing of cell-free DNA

Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

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