All samples were collected during planned bone marrow punctures of the iliac crest after disinfection of the skin and under sterile conditions using an aspiration cannula. Bone marrow punctures were scheduled in six patients for initial diagnosis of ALL, for bone marrow donation in four healthy participants and for other reasons in two participants (diagnostic purpose in one patient with severe aplastic anemia (SAA) and gathering of an autologous backup ahead of bone marrow transplantation in one patient suffering from sickle cell anemia). All work was conducted within the formal approval of the institutional ethics comittee (University Tuebingen, approval number 561/2020BO1, gathered 11/2020) and in accordance with the Declaration of Helsinki. Blood and bone marrow samples were collected after obtaining written informed consent of participant’s guardians and informing study participants according to the corresponding age group.

The sample collection for this study was performed in the midst or the end of the bone marrow aspiration, not at the beginning, to minimize the risk of contamination through commensal bacteria of the skin. A total of five milliliters of bone marrow was drawn, plus one additional milliliter of bone marrow for microbial cultivation as contamination control. Five milliliters of peripheral blood were collected through a peripheral venous blood draw or a central venous catheter (Hickman line). In patients below three years of age, two milliliters of blood were taken. Collection of bone marrow and peripheral blood was performed simultaneously, whenever possible.

The collected sample material was placed in DNA stabilizer tubes (Streck Cell-free DNA BCT, CE-IVD, Streck, La Vista, NE, USA) and sent to the laboratory at room temperature. After centrifugation for separation of plasma and peripheral blood cells, the plasma was stored at -80°C until further processing. Bone marrow samples were treated in the same manner.

Blood samples and bone marrow samples were processed in a blinded manner and therefore processed identically. Cells were separated from the supernatant by centrifugation at 1,600 x g for 10 min at 4°C and the supernatant was transferred to a fresh reaction tube. Then, a second centrifugation step at 16,000 x g for 10 min at 4°C was performed, supernatants were again transferred, and aliquots were further stored at -80°C. Nucleic acid isolation, quality controls and library preparation were carried out as previously described (Grumaz et al., 2016). NGS was performed on a Illumina ^© NextSeq 550 instrument. Adequate positive and negative controls accompanied all laboratory and sequencing procedures. Raw sequencing data were subjected to various QC controls comprising PHRED-Score filtering, adapter trimming, complexity filtering as well as k-mer based classification and contamination screening. To pass the quality filter, read quality needed to surpass a PHRED score of 20 and achieve a minimal length of 50 bp after quality control. All data generated were analyzed using Noscendo’s DISQVER platform, integrating the CE-IVD for pathogen detection assay from blood. The DISQVER platform comprises a curated microbial genome reference database of over 16,000 microbial species covering >1,500 pathogens and can detect bacteria, DNA-viruses, fungi, and parasites while differentiating contaminating commensals from infective agents via run controls and dedicated control collectives. For each pathogen type (bacterium, virus, fungus, parasite) a dedicated minimum threshold for reporting was set, that is further refined by incorporating data for each species individually regarding their observation in control cohorts. The limit of detection (LOD) was determined by bioinformatic and wetlab analytical validation procedures, which are further described in Supplementary Material 1 . For the PCA analysis, raw sequencing data after removal of human sequences and after classification of the non-human fraction was used for each sample. Read counts were log10 transformed prior to PCA analysis. The positive control was used after bioinformatic exclusion of the known, spiked-in microbial sequences, in order to not bias the results by taking these into account.

Statistical analyses were conducted using SPSS 27 (Armonk, New York) and RStudio 2022.12.0 (Boston, MA). Descriptive statistics are reported as median [range], if not otherwise specified. For comparison of means between cfDNA levels in control and ALL cohorts, the Wilcoxon rank sum test was used as values were not-normally distributed. For comparison of read mapping proportions between the different groups, the Kruskal-Wallis test was used.

As shown in Table 1 , participants were well balanced concerning gender and age. Common type ALL was the most frequent diagnosis in the ALL group, whereas controls were mainly healthy children and young adolescents willing to donate bone marrow for a sibling.

In the ALL group, four out of six patients received antibiotic treatment ahead of BMA. In three patients, intravenous Piperacillin/Tazobactam or Cefuroxime plus Clindamycin was administered just one day ahead of BMA. The remaining case with fever and mild pancytopenia was treated intravenously for six days with Cefuroxime and then discharged. Fourteen days later, ALL was diagnosed via BMA.

In the control group, only the two patients suffering from SAA or sickle cell anemia received antibiotics ahead of BMA: Piperacillin/Tazobactam in the SAA patient two days ahead of BMA, and long-term Penicillin prophylaxis in the sickle cell patient.

In total, an mean of 4.8 ng/µl of cell free DNA (cfDNA) could be isolated in the bone marrow of the ALL patients, compared to 2 ng/µl in the control group ( Table 1 , p=0.18). In peripheral blood, mean cfDNA concentration was 2.1 ng/µl in ALL and 0.3 ng/µl in the control group (p=0.08).

The cfDNA samples were processed in 6 different batches with different reagent lots due to logistical requirements. The batches separate into different clusters ( Figure 1A ) in contrast to the plot showing the different groups (patients, controls, synthetic sequencing run controls, Figure 1B ) where no separation based on any of these groups was possible. In the read mapping statistics regarding the proportion of human reads versus non-human reads ( Table 2 ), no significant differences between the groups were discernible ( Figure 2A ), with the exception of the synthetic control that shows a statistically significant increase in the non-human proportion, due to the synthetic microbial sequences ( Figure 2B ). In blood and bone marrow samples of patients or controls, no significant amounts of viral DNA were identified. DNA fragments for Torque Teno virus were found in two patients in the raw data, but below LOD and reporting thresholds.