Glycogen

The DNA of the selected specimen was enriched in P. jirovecii DNA using the NEBNext microbiome DNA enrichment kit based on the absence of CpG methylation (Biolabs), purified by ethanol precipitation in the presence of 10 μg glycogen (Thermo Fisher Scientific), and resuspended in 50 μl of 1× Tris-EDTA (TE) buffer. This enrichment raised the proportion of P. jirovecii DNA from a few percent to ca. 55% as determined a posteriori by high-throughput sequencing. Because only small amounts of DNA are recoverable from a clinical specimen and in absence of an in vitro culture system, a sufficient amount of DNA for high-throughput PacBio sequencing was obtained by random amplification. Five microliters of DNA was randomly amplified in a 50-μl reaction using the Illustra GenomiPhi HY DNA amplification kit (GE Healthcare). This amplification proved to create artificial molecules made of inverted repeats of several kilobases, which were revealed by PacBio sequencing. The reads from these molecules were eliminated by bioinformatics (described below). DNA was then purified using the QIAamp DNA blood minikit (Qiagen) followed by ethanol precipitation in the presence of 10 μg glycogen. Amplified DNA fragments were sized (mean of 8.6 kb) and quantified using a fragment analyzer (Advanced Analytical).

Trizol LS was used for RNA extraction from 400 μl plasma (Ambion, Life Technologies) as described previously (Papadaki et al., 2018 (link)). Briefly, following denaturation by Trizol LS, 25 fmoles of the synthetic C. elegans miRNA, cel-miR-39 (Qiagen GmbH, Hilden, Germany) were added in each sample to serve as an exogenous control. Chloroform was added for phase separation and after centrifugation, an equal volume of 700 μl of aqueous phase from each sample was precipitated by adding 0.7 volumes of isopropanol and 1 μl of glycogen (Qiagen). RNA pellet was resuspended in 50 μl RNAse-free water. RNA from all samples was kept at −80°C until further use in the subsequent cDNA synthesis step.

Total RNA from 400 μL plasma was extracted by Trizol LS (Ambion, Life Technologies, Waltham, MA, USA) as described previously [19 (link)]. Briefly, following denaturation by Trizol LS, 25 fmoles of the synthetic C. elegans miRNA, cel-miR-39 (Qiagen GmbH, Hilden, Germany), was added to each sample to serve as an exogenous control. After the addition of chloroform followed by centrifugation, an equal volume of 700 μL of aqueous phase from each sample was transferred to a clean eppendorf tube, and was precipitated by adding 0.7 volumes of isopropanol and 1 μL of glycogen (Qiagen, Hilden, Germany). RNA pellet was resuspended in 50 μL RNAse-free water. RNA from all samples was kept at −80 °C until further use in the subsequent cDNA synthesis step.