title: WGA of single Sphingomonadales bacterium cell sorted from coastal seawater by FACS
design: In brief: Each Single-Amplified-Genome (SAG) was obtained from a prokaryote-like particle that was FACS-isolated from coastal Gulf of Maine seawater. In depth: The seawater sample was incubated with the SYTO 9 nucleic acid stain (5 M final concentration; Thermo Fisher) on ice for 1060 min. Flow-cytometric analysis and sorting were performed using a BD InFlux Mariner flow cytometer equipped with a 488-nm laser for excitation and a 70-m nozzle orifice (Becton Dickinson, formerly Cytopeia). The cytometer was triggered on green fluorescence (531/40 BP emission filter). Sort gates for prokarya were defined on the basis of particle green fluorescence and forward scatter (Supplementary Fig. 4). Individual cells/particles were deposited into 384-well microplates containing 600 nl of 1 TE buffer per well and stored at 80 C until further processing.  Before genomic DNA amplification, sorted cells were lysed and their DNA was denatured by two freezethaw cycles and the addition of 700 nl of a lysis buffer consisting of 0.4 M KOH, 10 mM EDTA and 100 mM dithiothreitol, and a subsequent 10-min incubation at 20 C. The lysis was terminated by the addition of 700 nl of 1 M Tris-HCl, pH 4. Whole genome amplification was performed using WGA-X24. Libraries for sequencing of the amplified DNA were created with Nextera XT (Illumina) reagents following manufacturer instructions except for purification steps, which were done with column cleanup kits (QIAGEN QIAQuick PCR Purification and MinElute PCR Purification kits), and library size selection, which was done using BluePippin (Sage Science) with a target size of 500  50 bp. Libraries were sequenced with NextSeq 2000 (Illumina) and P3 reagents in 2  100 bp mode. Sequencing reads were demultiplexed with bcl2fastq2 v.2.2.0 software (Illumina) with the parameter barcode-mismatches=0. The demultiplexed reads were quality trimmed with Trimmomatic (v.0.32)54 using the following settings: -phred33 LEADING:0 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:36, then they were de-interleaved.