Journal: bioRxiv

Article Title: An optimized flow cytometry sorting-sequencing workflow reduces storage, sorting and extraction bias in sorted microbial communities

doi: 10.1101/2025.11.13.688239

Figure Lengend Snippet: Samples were sequenced with PacBio full-length 16S rRNA gene sequencing. The low biomass was obtained through dilution of the high biomass mock standard (10 7 cells mL -1 ) to 10 5 cells mL -1 before (diluted pre-extraction) and after (diluted post-extraction) DNA extraction and through sorting of the mock standard into 10 5 cells mL -1 and 10 5 noise events mL -1 using a non-selective SYBR® Green I staining. Dilution and sorting were performed in triplicate. FACS sheath fluid and aPBS diluent blanks were included in duplicate to identify and track the FACSome contaminants introduced during FACS-sequencing.

Article Snippet: The sorted cell fraction consisted of 288,000 cells collected in 1 mL FACS sheath fluid in 1.5 mL Eppendorf® DNA LoBind collection tubes (Merck, Darmstadt, Germany).

Techniques: Sequencing, Extraction, DNA Extraction, SYBR Green Assay, Staining

Journal: bioRxiv

Article Title: An optimized flow cytometry sorting-sequencing workflow reduces storage, sorting and extraction bias in sorted microbial communities

doi: 10.1101/2025.11.13.688239

Figure Lengend Snippet: The removal of relic DNA during sorting resulted in discrepancies between the non-sorted and sorted samples. Samples were sequenced with PacBio full-length 16S rRNA gene sequencing. The low biomass was obtained through dilution of the high biomass mock standard (10 7 cells mL -1 ) to 10 5 cells mL -1 before (diluted pre-extraction) and after (diluted post-extraction) DNA extraction and through sorting of the mock standard into 10 5 cells mL -1 and 10 5 noise events mL -1 using a non-selective SYBR® Green I staining. Dilution and sorting were performed in triplicate. FACS sheath fluid and aPBS diluent blanks were included in duplicate to identify and track the FACSome contaminants introduced during FACS-sequencing.

Article Snippet: The sorted cell fraction consisted of 288,000 cells collected in 1 mL FACS sheath fluid in 1.5 mL Eppendorf® DNA LoBind collection tubes (Merck, Darmstadt, Germany).

Techniques: Sequencing, Extraction, DNA Extraction, SYBR Green Assay, Staining

Journal: bioRxiv

Article Title: An optimized flow cytometry sorting-sequencing workflow reduces storage, sorting and extraction bias in sorted microbial communities

doi: 10.1101/2025.11.13.688239

Figure Lengend Snippet: Low biomass samples obtained through dilution of the high biomass mock standard (orange; 10 7 cells mL -1 ) to 10 5 cells mL -1 before (diluted pre-extraction; turquoise) and after (diluted post-extraction; green) DNA extraction and sorted noise events (10 5 mL -1 ; purple) did not display an improved clustering. Samples were sequenced with PacBio full-length 16S rRNA gene sequencing. Dilution and sorting with a non-selective SYBR® Green I cell staining were performed in triplicate. FACS sheath fluid (light grey) and aPBS (brown) diluent blanks were included in duplicate to identify and track the FACSome contaminants introduced during FACS-sequencing.

Article Snippet: The sorted cell fraction consisted of 288,000 cells collected in 1 mL FACS sheath fluid in 1.5 mL Eppendorf® DNA LoBind collection tubes (Merck, Darmstadt, Germany).

Techniques: Extraction, DNA Extraction, Sequencing, SYBR Green Assay, Staining

Journal: bioRxiv

Article Title: An optimized flow cytometry sorting-sequencing workflow reduces storage, sorting and extraction bias in sorted microbial communities

doi: 10.1101/2025.11.13.688239

Figure Lengend Snippet: Benzonase nuclease activity selectively depleted relic DNA of Veillonella atypica , Pseudomonas aeruginosa ASV63, Escherichia - Shigella coli ASV 11/42/43/77/88/179 and Salmonella enterica ASV 25/201. AMPure XP clean-up eliminated relic DNA but was not selective resulting in a distorted community composition. A decrease in the (relic) DNA concentration prior to sorting resulted in an inflation in absolute and relative contaminant sequencing read counts attesting the need of in silico data decontamination. Samples were sequenced with PacBio full-length 16S rRNA gene sequencing. Cells were sorted using a non-selective SYBR® Green I staining. A FACS sheath fluid blank was included to identify and track the FACSome contaminants introduced during FACS-sequencing.

Article Snippet: The sorted cell fraction consisted of 288,000 cells collected in 1 mL FACS sheath fluid in 1.5 mL Eppendorf® DNA LoBind collection tubes (Merck, Darmstadt, Germany).

Techniques: Activity Assay, Concentration Assay, Sequencing, In Silico, SYBR Green Assay, Staining