Cell-Free DNA Blood Collection Tubes Crosslinking Cellular DNA Impeding Nanopore Long-Read Sequencing.
Author(s): Chrysanthou S, Karmacharya T, Li J, Lu C, Cobbs CC, Mohibullah N
Publication: J Biomol Tech, 2025, Vol. 36, Page
PubMed ID: 40329983 PubMed Review Paper? No
Purpose of Paper
This paper compared DNA fragment length and Nanopore long read sequencing metrics from buffy coats collected in EDTA and Streck tubes and OCT-preserved tumor (unspecified type) specimens. In a follow-up study, the authors compared the same metrics using DNA from buffy coats collected in Streck tubes, EDTA tubes, and EDTA tubes with formaldehyde. The effect of adding a decrosslinking step to the extraction was also investigated.
Conclusion of Paper
In an initial study, significantly less sequencing data was obtained from blood collected in Streck tubes than blood collected in EDTA tubes or OCT-tissue specimens (90 GB versus ~116 GB and ~116 GB, respectively; P<0.001, both). The reduced sequencing data corresponded to lower estimated coverage in the Streck blood specimen than the EDTA blood specimen and OCT tissue specimen (~15% versus 47-48%). The post-shear length of DNA was longer from Streck blood specimen than EDTA blood (>60 kb in four of five specimens versus 25.1-34.3 kb); while not significant, the N50 pore activity was non-significantly lower in Streck blood than from EDTA blood. The authors postulate that the low data yield, longer shear length and lower pore activity could be attributed to DNA crosslinking by the formaldehyde in Streck tubes.
To further explore whether formaldehyde crosslinking interferes with sequencing and evaluate if decrosslinking could mitigate the effects, the authors tested the effects of adding formaldehyde to EDTA blood and including a decrosslinking step. When formaldehyde was added to EDTA blood the average shear length was >50 kb versus the 19.7-31.8 and 18.1- 25 kb in case-matched Streck and non-fixed EDTA blood, respectively. Additionally, the library yield was lower from EDTA blood specimens with formaldehyde (34-54 fmol) and Streck blood (60-131 fmol) than matched EDTA specimens without formaldehyde (120-160 fmol). DNA from Streck blood and formaldehyde-fixed EDTA blood also had a significantly lower N50 pore activity than DNA from unfixed EDTA blood (P<0.01). When a decrosslinking step (incubation in Nuclei Lysis Solutionat 56°C for 10 min at 2000 rpm, followed by an overnight incubation at 65°C) was added to the extraction, the average post-shear length, library yield and N50 pore activity of DNA from Streck blood and formaldehyde-fixed EDTA blood were comparable to that of unfixed EDTA blood (with or without the additional step). Further, adding the decrosslinking step improved the oxidation error rate.
Studies
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Study Purpose
This study compared DNA fragment length and Nanopore long read sequencing metrics from buffy coats collected in EDTA and Streck tubes and OCT-preserved tumor (unspecified type) specimens. In a follow-up study, the authors compared the same metrics using DNA from buffy coats of blood collected in Streck tubes, EDTA tubes, and EDTA tubes with formaldehyde. The effect of adding a decrosslinking step to the extraction was also investigated. Sequencing data obtained from Streck blood, EDTA blood and OCT-embedded tumor from five patients were used in an initial analysis (no further details were included). For this study, blood was collected from three healthy volunteers into one Streck cfDNA BCT and two BD Vacutainer EDTA tubes. Formaldehyde was added to one of the EDTA tubes to achieve a concentration of 0.1%. Blood was stored for 20 h before separation of buffy coat by centrifugation at 800 g for 10 min. Each buffy coat specimen was divided into two specimens prior to DNA extraction. One specimen of each pair was subjected to decrosslinking consisting of the addition of Nuclei Lysis Solution, incubation at 56°C for 10 min at 2000 rpm, and overnight incubation at 65°C before leukocyte lysis. DNA was extracted from untreated and de-crosslinked buffy coats in phosphate buffered saline using the Monarch HMW DNA Extraction Kit for Cells & Blood with the addition of a decrosslinking step for one specimen from each buffy coat. DNA was quantified using the Quant-iT 1X dsDNA High Sensitivity Assay Kit and NanoDrop spectrophotometry. DNA size was analyzed by TapeStation 4200. DNA was sheared to produce 25 kb fragments using a g-tube at 4600-6000 rpm for 2-4 min. Sequencing libraries were prepared using the Oxford Nanopore Ligation Sequencing Kit and sequenced on a FLO-PRO114M.
Summary of Findings:
In an initial study, significantly less sequencing data was obtained from blood collected in Streck tubes than blood collected in EDTA tubes or OCT-tissue specimens (90 GB versus ~116 GB and ~116 GB, respectively; P<0.001, both). The reduced sequencing data corresponded to lower estimated coverage in the Streck blood specimen than the EDTA blood specimen and OCT tissue specimen (~15% versus 47-48%). The post-shear length of DNA was longer from Streck blood specimen than EDTA blood (>60 kb in four of five specimens versus 25.1-34.3 kb); while not significant, the N50 pore activity was non-significantly lower in Streck blood than from EDTA blood. The authors postulate that the low data yield, longer shear length and lower pore activity could be attributed to DNA crosslinking by the formaldehyde in Streck tubes.
To further explore whether formaldehyde crosslinking interferes with sequencing and evaluate if decrosslinking could mitigate the effects, the authors tested the effects of adding formaldehyde to EDTA blood and including a decrosslinking step. When formaldehyde was added to EDTA blood the average shear length was >50 kb versus the 19.7-31.8 and 18.1- 25 kb in case-matched Streck and non-fixed EDTA blood, respectively. Additionally, the library yield was lower from EDTA blood specimens with formaldehyde (34-54 fmol) and Streck blood (60-131 fmol) than matched EDTA specimens without formaldehyde (120-160 fmol). DNA from Streck blood and formaldehyde-fixed EDTA blood also had a significantly lower N50 pore activity than DNA from unfixed EDTA blood (P<0.01). When a decrosslinking step (incubation in Nuclei Lysis Solution at 56°C for 10 min at 2000 rpm, followed by an overnight incubation at 65°C) was added to the extraction, the average post-shear length, library yield and N50 pore activity of DNA from Streck blood and formaldehyde-fixed EDTA blood were comparable to that of unfixed EDTA blood (with or without the additional step). Further, adding the decrosslinking step improved the oxidation error rate.
Biospecimens
Preservative Types
- Streck/BCT
- Formalin
- None (Fresh)
Diagnoses:
- Neoplastic - Not specified
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Decrosslinking performed
No decrosslinking performed
Biospecimen Acquisition Type of collection container/solution EDTA tube
Streck tube