D1000 screentape system

The 16S ribosomal RNA gene amplicons were prepared for the Illumina MiSeq System according to the manufacturer’s protocol as was previously described [19 (link)]. Briefly, libraries were prepared using two PCR steps. In the first PCR step, the V3 and V4 regions of the 16S rRNA gene (460 bp) were amplified using 25 cycles with 5 μL of the extracted DNA. The full-length primer sequences, using standard IUPAC nucleotide nomenclature, to follow the Illumina protocol targeting this region were: 16S Amplicon PCR Forward Primer = 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and the Reverse Primer = 5′ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. The PCR products were visualized using TapeStation System (Agilent, Santa Clara, CA, USA) with D1000 ScreenTape System (Agilent, Santa Clara, CA, USA). The PCR products were purified using AMPure XP beads (Beckman Coulter, CA, USA). In the second PCR step Nextera XT Index kit (Ilumina) primers were used to label the first PCR product using 8 cycles. The second PCR products were purified using AMPure XP beads. All purified PCR products were pooled together and normalized to 10 nM. The 10 nM stock was diluted to 4 nM, which was denaturated using NaOH and 20% PhiX control. The libraries (10pM) were then sequenced in the Illumina Miseq (Ilumina, San Diego, CA, USA) using the V2 kit for 500 cycles (250x2, paired end reads).

Triplicates of each barcoded amplicon sample were combined. Each sample was diluted × 10⁵–10⁷-fold and the molar concentration of barcoded amplicons was quantified using a home-brew ddPCR library quantification assay and KAPA SYBR FAST Universal qPCR Library Quantification Kit (#KK4824, Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s instructions (the qPCR reaction was set up same as above).
Barcoded samples were pooled in equimolar amounts. The pooled library was purified using Agencourt AMPure XP beads (#A63880, Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions and eluted with ultrapure water (Invitrogen).
The purified library was confirmed to have the 260 nm to 280 nm light absorbance ratio of > 1.8 using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). The average amplicon size of approximately ~ 400 bp was confirmed with a High Sensitivity D1000 ScreenTape System (#5067-5584 and #5067-5585, Agilent Technologies, Santa Clara, CA, USA) using a 2200 TapeStation instrument (Agilent Technologies) and the Agilent 2200 TapeStation Software A.02.01 (Agilent Technologies).
The molar concentration of the pooled library was measured using the ddPCR and KAPA qPCR assays, and the library was submitted for NGS with the sequencing primers described in Additional file 1: Table S1.

Total DNA was extracted by the phenol-chloroform method [41 (link)]. Library preparation was performed using the Nextera DNA Flex Library Prep (Illumina) kit. DNA concentration and quantification were assessed using QuBit 4 with the DNA High Sensitivity test. Fragment size was visualized using Tapestation (Agilent Technologies) with the D1000 ScreenTape System. Genomic DNA was sequenced with the Illumina NextSeq 550. The quality of the read libraries was assessed using the FastQC v0.11.9 tool [42 ]. Pre-processing was performed with the Trim Galore v0.6.7 [43 ]. Genome assembly was conducted with SPAdes v. 3.15.4 [44 (link)]. Gene prediction was performed with Prokka v1.14.15 [45 (link)] and the genome completeness was calculated with the CheckM v1.1.11 tool [46 (link)]. The complete genome of strain 34SFC-3 is deposited at NCBI under the BioProject PRJNA894989.