Nextera barcode

Microbial genomic DNA was extracted from HRS and DRS samples using the FastDNA Spin Kit (MP Biomedicals, Irvine, CA, USA) and quantified using Epoch Spectrometer (Biotek, VT, USA). PCR amplification was performed using primers targeting V3 and V4 regions of 16S rRNA genes. The first round of amplification was carried out using primers 341F and 805R (Table S1) under the following conditions: denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 5 min. Secondary amplification was performed to attach the Illumina NexTera barcodes using primers i5-F and i7-R (Table S1) under the same amplification conditions as described above; however, the number of amplification cycles was set to eight. The PCR products were separated by electrophoresis on 1% agarose gel and visualized using a Gel-Doc system (Bio-Rad, Hercules, CA, USA). Then, the PCR products were purified using the CleanPCR Kit (CleanNA, Waddinxveen, the Netherlands), and equal concentrations of the purified products were pooled together. Nontarget short fragments were removed using the CleanPCR Kit, and the quality and size of PCR products were assessed using the DNA 7500 chip on Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). Pooled amplicons were sequenced at ChunLab, Inc. (Seoul, South Korea) using the Illumina MiSeq platform, according to the manufacturer’s instructions.

The composition of newborn microbiota was analyzed using 16S rRNA amplicon sequencing with Illumina MiSeq (Illumina, Inc., San Diego, CA, USA). For sequencing, the V3-V4 regions of the bacterial 16S rRNA gene were amplified using primer set F319 (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and R806 (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGG TATCTAATCC-3′). DNA templates (12.5 ng/µL) were amplified using a KAPA HiFi Hotstart PCR Kit (Kapa Biosystems, Kenilworth, NJ, USA) with 5 µM of primers. Reaction conditions were as follows: 95 °C for 3 min; 25 cycles of 95 °C for 30 s; 55 °C for 30 s; and 72 °C for 30 s, with a final extension at 72 °C for 5 min. After PCR cleanup, a secondary amplification to attach Illumina Nextera barcodes was performed using i5 forward and i7 reverse primers. The DNA was amplified according to the manufacturer’s protocol. The PCR products were purified using an Agencourt AMpure XP PCR Purification Kit (Beckman Coulter, High Wycombe, UK). The purified products were quantified using a QuantiFluor^® ONE dsDNA System (Promega, Madison, WI, USA). The products’ size and quality were evaluated on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The pooled libraries were sequenced using an Illumina MiSeq instrument with a MiSeq v3 Reagent Kit (Illumina, Inc., San Diego, CA, USA).

Amplification of the bacterial 16S rRNA gene’s V3–V4 variable region was achieved through a biphasic PCR method for 25 cycles at 55 °C. In summary, the PCR utilized a set of two primers: the forward primer was 5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACG-GGNGGCWGCAG and the reverse primer was 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA-GGACTACHVGGGTATC-TAATCC-3′. The PCR products underwent evaluation using 2% agarose gel electrophoresis. The 16S rRNA libraries were then purified with magnetic beads (AMPure XP), in line with the specifications given by Beckman Coulter, Wycombe, UK. To verify the purity of the samples, a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) was employed. The second phase of the PCR involved attaching Illumina Nextera barcodes to the products of the initial PCR using i5 forward and i7 reverse primers. These amplified products were then purified following the same procedure as the initial round. DNA quantification was carried out using the QuantiFluor® ONE dsDNA System (Promega), and the Bioanalyzer 2100 was again used for assessing the quality of the samples. The amplified 16S rRNA gene and the prepared library, developed using this two-step PCR method, were sequenced using the MiSeq v3 Reagent Kit from Illumina, Inc.

The DNA from bulk soil or rhizosphere soil was extracted using the ISOIL for beads beating DNA extraction kit (Nippongene, Japan) following the manufacturer’s instructions. Extracted DNA was stored at – 20 °C prior to PCR amplification. For bacterial microbial community profiling, the V4 region of the 16S rRNA gene was amplified using the universal primers: 515F^{54 (link)} (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GTGCCAGCMGCCGCGGTAA-3’) and 806R^{55 (link)} (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GGACTACHVGGGTWTCTAAT-3’). For fungal community structure analysis, universal primer pairs for ITS1 spacer region were used: ITS1F (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GTCTCGTGGGCTCGGAGATGTGTATA AGAGACAG-GCTGCGTTCTTCATCGATGC-3’) for ITS1 amplification^{56 (link)}.
PCRs were performed using AmpliTaq GOLD (Applied Biosystems; Carlsbad, CA, USA) and PCR conditions were as follows: (1) 95 °C for 10 min, (2) 95 °C for 30 s, (3) 55 °C for 30  s, (4) 72 °C for 1 min and (5) 72 °C for 7 min, repeated (2)–(4) 30 cycles. Illumina sequencing library preparation was performed as per the Illumina, 2013, Illumina Co., California, USA, using Nextera barcodes. Prepared libraries were sequenced by Macrogen Co. (Seoul, Korea) using the MiSeq platform (Illumina Co., California, USA). Sequence data was returned as demultiplexed fastq paired-end files.

The V3 to V4 variable region of the 16S rRNA gene was amplified using previously described primers [31 (link)]. Library preparation followed guidelines from Illumina. PCR was performed using the KAPA HiFi HotStart ReadyMix kit (Kapa, Biosystems). The amplification procedure has been previously described. PCR products were purified with AMPure XP magnetic beads (Beckman, Coulter) and quantified using a Qubit fluorometer (Invitrogen, Life Technologies). Secondary PCR amplification was performed to add Illumina Nextera barcodes and the products we purified to remove nontarget fragments. Amplicons were normalized, pooled, and sequencing using an Illumina Miseq system (Illumina, San Diego, USA) [28 ].