Fig. 1

Main steps of 5WBF. From 50 to 400 µL of whole blood were diluted in RPMI 1640 medium or PBS 1× buffer in a large flask. The cartoon shows 200 µL of whole blood as an example. The diluted sample was loaded into a 10 mL syringe before the 5 μm filter was connected to the syringe. The blood was filtered by very gentle pressure (ideally, drop by drop) with the syringe plunger, until the plunger reached the bottom of the syringe to recover the maximum of infected RBCs. The filtration step itself is rapid and takes about 1 to 3 min. The filtrate was centrifuged at 2500g for 5 min and the supernatant was discarded. The pellet was suspended with ~ one pellet volume of RPMI 1640 or PBS 1×, transferred into a 1.5 mL tube, and stored until DNA extraction. (i) from the experiments, the filter dead volume was about 200 µL (reported as 100–150 µL by the manufacturer); (ii) even after the 2 mL optional wash with RPMI/PBS, the filter had a red colour indicating some retained RBCs or haemolysis during filtration; RBCs loss seems low although no precise quantification was done; (iii) the harder the push with the syringe plunger, the more haemolysis occurs; (iv) even with gentle push, some haemolysis can occur with some clinical samples and the filtrated pellet after centrifugation was slightly smaller, but WGS data were fine; (v) on some occasions, an air bubble could block the filter; then a slight flick at the bottom of the syringe (close to the filter) was applied; and (vi) for practical reason, a slightly different protocol was also tested in which the diluted blood sample was loaded into a 10 mL syringe after the 5 µm filter was connected to the syringe (Additional file 1: Fig. S1); similar results were obtained than with the protocol described in this Fig. 1