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Heat inactivation does not alter host plasma cell-free DNA characteristics in infectious disease research.

Author(s): Luo Y, Zhang H, Li L, Lin Y, Wang X, Chen W, Tao Y, Ou R, Zhou W, Zheng F, Jin Y, Cheng F, Zhu H, Zhang Y, Jin X

Publication: Clin Chim Acta, 2023, Vol. 553, Page 117751

PubMed ID: 38163539 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, size distribution, and sequence representation of cfDNA and cfmtDNA isolated from plasma specimens that were subjected to one or two rounds of heat inactivation relative to case-matched plasma that did experience heat activation. Blood was collected from seven healthy volunteers into EDTA tubes and plasma was obtained by centrifugation at 600 g for 10 min at 4°C within 4 h of collection. The supernatant was centrifuged again at 16,000 g for 10 min at 4°C and aliquoted. Two aliquots were subjected to heat inactivation consisting of incubation at 56°C for 30 min while the remaining aliquot was stored on ice for 30 min, after which all three aliquots were frozen at -80°C. A week later, one of the aliquots was thawed at room temperature before undergoing a second round of heat inactivation at 56°C for 30 min followed by storage at -80°C. DNA was extracted from plasma using the MagPure Circulating DNA KF Kit and yield was quantified using the Qubit dsDNA High Sensitivity Assay Kit. Sequencing libraries were prepared using the MGIEasy Cell-free DNA Library Prep Kit and pair-end 100 bp sequencing was performed on a DNBSEQ instrument. The size profile of the isolated cfDNA was calculated based on the distance between pairs of pair-end reads. Reads that were properly aligned to the mitochondrial genome were considered to be mtDNA. Motif analysis was conducted by comparing the first four bases at the 5’ end of the cfDNA.

   Summary of Findings:

   cfDNA yields were comparable among plasma specimens that was not subjected to heat inactivation and plasma specimens that were subjected to one or two rounds of heat inactivation at 56°C for 30 min. The size profiles of cfDNA isolated from plasma that were subjected to 0-2 rounds of heat inactivation overlapped almost completely. Further, no difference in the percentage of fragments that were between 60-155 bp or 170-250 bp was observed among plasma specimens that did or did not experience heat inactivation. Finally, z-scores that were calculated by comparing the mean and the standard deviation of each cfDNA size frequency did not differ between plasma specimens that were subjected to one or two rounds of heat inactivation and plasma specimen that were not heated.  Principal component analysis based on the 5’ end motif clustered specimens by volunteer not by heat inactivation status, and no differences were observed in the motif diversity score between heat inactivation groups. Correlations in the 5’ end motif were stronger between heat-inactivation groups than between specimens from different volunteers (P=1.7 e^-7). Measured genome representation was similar between heat inactivation groups in specimens from each of the seven volunteers. Further, heat inactivation did not affect window protection score patterns or the relative coverage of loci within 2000 bp of transcriptional start sites (TSS) or CCCTC-binding factor (CTCF) sites.

   Median mtcfDNA read counts were also comparable among plasma specimens regardless of heat inactivation. mtcfDNA size profiles were similar for mtcfDNA isolated from plasma that was not subjected to heat inactivation and plasma that was subjected to one or two rounds of heat inactivation, but when the percentage of fragments of each size were compared, there was slightly less cfmtDNA <80 bp and slightly more >100 bp when plasma was heat-inactivated; further, the effect appeared greater when two cycles of heat inactivation were applied rather than one cycle. Nevertheless, no difference in z-scores (which were calculated based on the mean and the standard deviation of each cfmtDNA size frequency) was observed between heat inactivation groups.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood processing method	Not heat inactivated Heat inactivated once at 56°C for 30 min Heat inactivated twice at 56°C for 30 min
   Analyte Extraction and Purification	Incubation duration/condition	No heat inactivation Heat inactivated once at 56°C for 30 min Heat inactivated twice at 56°C for 30 min

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