Shotgun plasma cfDNA sequencing in healthy and septic newborn foals
Sepsis and systemic inflammatory response syndrome (SIRS) due to a bacterial infection are primary causes of mortality in newborn foals. Improving our grasp of disease biology, enhancing diagnostic accuracy, and advancing treatment options are pivotal for boosting survival rates. To deepen our understanding of the pathophysiological mechanisms underlying sepsis and SIRS, we performed genome-wide shotgun plasma cell-free DNA (cfDNA) Illumina sequencing on blood plasma of 32 healthy and sick newborn (1-3 day old) foals. Subsequently, we used Kraken2 for taxonomic assignment, necessary for precise bacterial profiling and pathogen detection.  Overall design: in this study, we enrolled a total of 32 foals (F01-F32), including 25 sick foals admitted to the emergency clinic of the Utrecht University Equine Hospital in The Netherlands. According to neonatal SIRS criteria, 11 foals were SIRS-positive (S+; nSIRS = 3), 4 were SIRS-negative with no symptoms (nS-; nSIRS = 0), and 10 were SIRS-negative but exhibited one or multiple symptoms (sS-; nSIRS =1-2). Peripheral blood was collected from all foals using Streck Cell-Free BCT tubes. Plasma extraction was performed followed by cfDNA extraction using the Qiagen CNA kit and synthetic oligo were spiked in (fixed sequenced of 50bp, 100bp, and 150bp). Sequencing libraries were then prepared using the SRSLY PicoPlus NGS Library Prep Kit for Illumina, and subsequent sequencing was conducted on the NovaSeq 6000 platform with 2 x 150bp reads. To enable identification of potential DNA contaminants during sample handling, we incorporated four negative controls (NTC1, NTC2, NCMQiso, and NCMQlib). Beside we included three positive controls (PC1, PC2, and PC3) for taxonomic classification quality control. There positive controls constisted of sonicat4ed mock community DNA (ZymoBIOMICS Microbial Community DNA Standard) containing a mixture of genomic DNA of 10 microbial strains.