title: Microbiome sample from the blood of septic patient
design: Suppression adapters were generated from a long and short oligonucleotide (Sigma-Aldrich, Steinheim). Both oligonucleotides were incubated with 10 L 10x T4 ligase mix (New England Biolabs, Frankfurt) and 50 L nuclease-free water for 3 min at 95 C and cooled down overnight at RT. SUPSETS workflow starts with ligating suppression adapters to blunt-end DNA fragments (prepared cfDNA), followed by elongation in the presence of a panel of Fusion primers. For clinical samples, a defined volume of 15.92 L blunt-end cfDNA was used. After AMPure XP beads purification, second PCR amplification was performed using US-FW and Ad-rev primers (both Sigma-Aldrich, Steinheim). Purified amplification products were applied to Nanopore sequencing. For MinION Flongle experiments, the Ligation Sequencing Kit SQK-LSK 109 for sample preparation and the Native Barcoding Expansion Kit EXP-NBD 104 were used according to the manufacturers protocol. The clinical samples were sequenced for 24 h and FL_04 also alone for 3 h. The basecalling was done using guppy in high accuracy mode.