Tbs 380 mini fluorometer

Total DNA from rhizoplane soils and fibrous root segments was, respectively, extracted using the DNeasy Plant Mini Kit and the DNeasy PowerSoil Pro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. DNA quality and quantity were assessed using a TBS-380 Mini-Fluorometer (Turner Biosystems, CA, USA) and a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), respectively.

Total bacterial DNA of stool samples were extracted using the TIANamp Stool DNA Kit (Tiangen Biotech, Beijing, China; cat number: DP328) following the manufacturer’s instructions. The concentrations and purity of DNA were measured by a Turner TBS‐380 mini‐fluorometer (Turner Biosystems, Sunnyvale, CA, USA) and a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The extracted DNA was then stored at -20°C. The genomic DNA quality was further evaluated by 1% agarose gel electrophoresis before metagenomic sequencing.

DNA for metagenomic analysis was extracted from approximately 0.5 g soil samples using the fastDNA Spin Kit (MP Biomedicals, Solon, OH, USA). The concentration and purity of extracted DNA were determined using a TBS-380 mini-Fluorometer (Turner Biosystems, Sunnyvale, CA, USA) and an ND-2000 UV-Vis spectrophotometer (Thermo Fisher Scientific-NanoDrop, Wilmington, DE, USA), respectively. Extracted DNA was assessed using 1% agarose gel electrophoresis. DNA was sheared into fragments of approximately 400 bp using an M220 Focused-ultrasonicator (Covaris, Woburn, MA, USA). DNA templates were then treated using the Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA) to build a paired-end library. Pair-end sequencing (2 × 150 bp) was completed on the Novaseq platform (Illumina, San Diego, CA, USA) by Shanghai Majorbio Biopharm Technology Co., Ltd. in Shanghai, China.

Total DNA was extracted from filters using a FastDNA Spin Kit (mBio, USA) according to the manufacturer’s instructions. The concentration and purity of extracted DNA were determined with a TBS-380 Mini-Fluorometer (Turner Biosystems, Sunnyvale, CA, USA) and a NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), respectively. The DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTflex™ Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Paired-end sequencing was performed on an Illumina Hiseq instrument (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co. (Shanghai, China) using HiSeq Reagent kits according to the manufacturer’s instructions.¹ In total, 18 metagenomes (i.e., three biological replicates from six time points during algal succession) were sequenced.

The V1-V2 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers 27Fmod (5′-AGRGTTTGATYMTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′) as described previously^{33 (link)}. Amplicons generated from each sample (~ 330 bp) were subsequently purified using AMPure XP (Beckman Coulter). DNA was quantified using a Quant-iT Picogreen dsDNA assay kit (Invitrogen) and a TBS-380 Mini-Fluorometer (Turner Biosystems). The 16S rRNA gene sequencing was performed with the MiSeq sequencing system (Illumina, San Diego, CA, USA) according to Illumina protocol.

Concentration and purity of the genomic DNA were quantified with TBS-380 Mini-Fluorometer (Turner BioSystems) and NanoDrop 2500, respectively. The genome of strain Cp2 was sequenced by Shanghai Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China using Illumina HiSeq and PacBio methods. After sequencing, clean reads were obtained from the raw reads by removing adaptor sequences, reads containing low-quality reads and poly-N. The resulting clean reads were used for scaffolding by SOAPdenovo software. High-quality PacBio long reads were assembled by using Canu (Koren et al., 2017 (link)). After assembly, circular chromosomes and plasmids with gapless were finally generated and drawn using the CGView program (Stothard and Wishart, 2005 (link)). Prediction of genes for protein coding sequences (CDSs), transfer RNA (tRNA), and ribosomal RNA (rRNA) was performed with Glimmer, GeneMarks, tRNAscan-SE, and Barrnap software, respectively (Besemer et al., 2001 (link); Seemann, 2013 ; Lowe and Chan, 2016 (link)). In addition, each predicted CDS was annotated through the Clusters of Orthologous Genes (COG) database by BLAST (Tatusov et al., 2003 (link)). Genes associated with virulence were identified in the Virulence Factors Database (VFDB) and Pathogen-Host Interaction Database (PHI-base) (Chen et al., 2005 (link); Winnenburg et al., 2006 (link)).