Effects of blood-processing protocols on cell-free DNA fragmentomics in plasma: Comparisons of one- and two-step centrifugations

Xintao Hu; Haiqiang Zhang; Yeqin Wang; Yu Lin; Qiuyan Li; Lingguo Li; Guodan Zeng; Rijing Ou; Xinyu Cheng; Yan Zhang; Xin Jin

doi:10.1016/j.cca.2024.119729

Effects of blood-processing protocols on cell-free DNA fragmentomics in plasma: Comparisons of one- and two-step centrifugations

Clin Chim Acta. 2024 May 14:560:119729. doi: 10.1016/j.cca.2024.119729. Online ahead of print.

Authors

Xintao Hu¹, Haiqiang Zhang², Yeqin Wang², Yu Lin², Qiuyan Li², Lingguo Li², Guodan Zeng², Rijing Ou², Xinyu Cheng³, Yan Zhang⁴, Xin Jin⁵

Affiliations

¹ Engineering Research Center of Text Computing & Cognitive Intelligence, Ministry of Education, Key Laboratory of Intelligent Medical Image Analysis and Precise Diagnosis of Guizhou Province, State Key Laboratory of Public Big Data, College of Computer Science and Technology, Guizhou University, Guiyang 550025, China; BGI Research, Shenzhen 518083, China.
² BGI Research, Shenzhen 518083, China.
³ Engineering Research Center of Text Computing & Cognitive Intelligence, Ministry of Education, Key Laboratory of Intelligent Medical Image Analysis and Precise Diagnosis of Guizhou Province, State Key Laboratory of Public Big Data, College of Computer Science and Technology, Guizhou University, Guiyang 550025, China.
⁴ BGI Research, Shenzhen 518083, China. Electronic address: zhangyan15@genomics.cn.
⁵ BGI Research, Shenzhen 518083, China; School of Medicine, South China University of Technology, Guangzhou 510006, China. Electronic address: jinxin@genomics.cn.

PMID: 38754575
DOI: 10.1016/j.cca.2024.119729

Abstract

Background: Cell-free DNA (cfDNA) fragmentomic characteristics are promising analytes with abundant physiological signals for non-invasive disease diagnosis and monitoring. Previous studies on plasma cfDNA fragmentomics commonly employed a two-step centrifugation process for removing cell debris, involving a low-speed centrifugation followed by a high-speed centrifugation. However, the effects of centrifugation conditions on the analysis of cfDNA fragmentome remain uncertain.

Methods: We collected blood samples from 10 healthy individuals and divided each sample into two aliquots for plasma preparation with one- and two-step centrifugation processes. We performed whole genome sequencing (WGS) of the plasma cfDNA in the two groups and comprehensively compared the cfDNA fragmentomic features. Additionally, we reanalyzed the fragmentomic features of cfDNA from 16 healthy individuals and 16 COVID-19 patients, processed through one- and two-step centrifugation in our previous study, to investigate the impact of centrifugation on disease signals.

Results: Our results showed that there were no significant differences observed in the characteristics of nuclear cfDNA, including size, motif diversity score (MDS) of end motifs, and genome distribution, between plasma samples treated with one- and two-step centrifugation. The cfDNA size shortening in COVID-19 patients was observed in plasma samples with one- and two-step centrifugation methods. However, we observed a significantly higher relative abundance and longer size of cell-free mitochondrial DNA (mtDNA) in the one-step samples compared to the two-step samples. This difference in mtDNA caused by the one- and two-step centrifugation methods surpasses the pathological difference between COVID-19 patients and healthy individuals.

Conclusions: Our findings indicate that one-step low-speed centrifugation is a simple and potentially suitable method for analyzing nuclear cfDNA fragmentation characteristics. These results offer valuable guidance for cfDNA research in various clinical scenarios.

Keywords: Cell-free DNA; Centrifugation; Fragmentomics; Plasma DNA.