Additional file 1 of A pipeline for targeted metagenomics of environmental bacteria

Additional file 1: Figure S1. The development of our pipeline was done in three steps before the application on an environmental sample. (1) In the first step, four bacterial isolates (GC content is given in mol%) were treated with four different fixatives (plus unfixed control) and their signal intensity was measured by flow cytometry (1a). Glyoxal was not further analyzed due to low signal intensities (red cross). Three isolates and three of the brighest fixations (plus control) were sorted (100 and 500 cells) and forwarded to MDA (1b). The MDA products of two isolates were sequenced and their genome quality assessed (1c). The best results in total (signal intensity and genome quality) were achieved with ethanol fixation and 500 cells (green star). (2) In the second step, the HCR-FISH protocol from Yamaguchi et al. [21] was adapted with different denaturation temperatures, hybridization buffers and amplification times. The signal intensities were assessed after HCR-FISH via microscopy. (3) In a third step, the optimized HCR-FISH protocol was tested for validation on isolates with ethanol fixation (plus unfixed control). There were no significant differences in assembly metrics between ethanol fixation and unfixed control samples after sequencing of MDA products from 500 sorted cells. (4) The optimized HCR-FISH protocol was tested on ethanol fixed and unfixed seawater samples. Bacteroidetes and the flavobacterial clade Vis6 were targeted by specific HCR-FISH probes, 500 cells sorted and sequenced. For comparison a whole community shotgun metagenome was prepared.