Miseq sequencing platform

The V3-V4 hypervariable region of 16S rRNA genes were amplified using universal primers with overhang adapters (338 Fin TTCCCTACACGACGCTCTTCCGATCT-ACTCCTACGGRAGGCAGCAG; 806 Rin GAGTTCCTTGGCACCCGAGAATTCCA-GGACTACHVGGGTWTCTAAT) by the Phusion High Fidelity PCR Master Mix with HF Buffer (New England Biolabs, Hitchin Herts, UK). The PCR reactions were performed as follows: initial denaturation at 94 °C × 2 min; 25 cycles of denaturation at 94 °C × 30 s, annealing at 56 °C × 30 s, elongation at 72 °C × 45 s; and final extension at 72 °C × 2 min and held at 10 °C × 10 min. After purification using the AXYGEN AxyPrep DNA Gel Extraction Kit (Axygen Scientific, Union City, CA, USA), the libraries were then normalized according to Qubit3.0. The barcoded 16S rRNA gene was sequenced on the Illumina MiSeq sequencing platform (Illumnia, Inc., San Diego, CA, USA) at TinyGene Bio-Tech (Shanghai), Co., Ltd. (Shanghai, China), using a 2 × 300 cycle V3 kit, following standard Illumina sequencing protocols.

DNA extraction was performed during the stable phase using a Fast DNA™ SPIN Kit for Soil (MP Biomedicals, Canada), after which the extracted DNA was sequenced using the Illumina MiSeq sequencing platform (Novogene Co., Beijing, China) and the 515F (5′-GTGCCAGCAGCCGCGGTAA-3′) and 806R (5′-GGACTACCAGGGTATCTAAT-3′) targeting the V4 region of the 16S rRNA gene. Each reverse primer contained a 6-bp error-correcting unique barcode and PCR amplification was conducted by subjecting the samples to 98 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s, and then final extension at 72 °C for 10 min. Sequences were then analyzed using the Illumina MiSeq platform (Novogene Co., Beijing, China).

MAP isolates were selected for WGS to 1) represent the approximate proportion of isolates per VNTR type in Canada (while minimizing duplicate types from the same herd), 2) include all Canadian provinces currently represented in the MAP collection, and 3) represent three different scenarios of VNTR type diversity at the herd-level in Alberta. The three Alberta herd-level isolate sample sets included a herd with (A) four isolates of the same VNTR type, (B) three isolates with three different VNTR types, and (C) four isolates with two different VNTR types. As these isolates were cultured from environmental manure samples, true herd-level diversity was not assessed.
MAP DNA was prepared for sequencing using the NexteraXT sample preparation kit (Illumina, San Diego, CA, USA). Samples were multiplexed to achieve paired end reads with an average coverage of 50X and sequenced using V2 (250 bp reads) or V3 (300 bp reads) chemistry using the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA).

The maize rhizosphere soil from normal conditions and medium drought treatment on the 26th day after inoculation were collected and used for high-throughput sequencing. After extracting the DNA of each sample, it was subjected to 1% agarose gel electrophoresis. Specific primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with barcodes were used to amplify 16S rRNA of the bacterial V3-V4 region. The PCR products were detected and quantified by QuantiFluor™-ST Blue Fluorescence Quantification System (Promega), and then, each sample was mixed in the corresponding proportion. The following thermal program was used for amplification: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 45 s (a total of 30 cycles), and finally, extension at 72°C for 10 min. The reaction products were detected using 2% agarose gel electrophoresis. The Miseq library was constructed with TruSeq™ DNA Sample Prep Kit reagents, and the data were optimized using Trimmomatic and FLASH software after sequencing was completed on the Illumina MiSeq sequencing platform (Majorbio, Shanghai, China).

Total genomic DNA from the soil was extracted using a Mobio PowerSoil^® DNA Isolation Kit (Mobio, USA). After the extraction of genomic DNA, 1% agarose gel electrophoresis was used to detect the extracted genomic DNA. Using extracted soil genomic DNA as the template, the 16S rDNA v3-v4 region was selected as the amplified fragment by PCR amplification, followed by high-throughput sequencing. Primer sequences were 336F (5'—gtactcctacgggaggcagca-3 ') and 806R (5'—gtggactachvgggtwtctaat-3 ') [35 (link)]. The PCR system (25 μL) involved the following: 30 ng DNA samples, forward primer (5 μmol/L) 1 μL, reverse primer (5 μmol/L) 1 μL, BSA (2 ng/μL) 3 μL, 2 x Taq Plus Master Mix 12.5 μL, and dd H₂O 7.5 μL. The PCR conditions were as follows: predenaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 60 s, and 30 cycles. Finally, the process was extended at 72°C for 7 min. The amplification results were subjected to 2% agarose gel electrophoresis, and the PCR products were recovered using an AxyPrepDNA gel recovery kit (AXYGEN). The PCR products were eluted by Tris_HCl, detected by 2% agarose electrophoresis and then sequenced by the Illumina Miseq sequencing platform from the Beijing Ollwegene Technology Co., LTD.

Whole genome sequencing of the confirmed Lactobacillus genus was done adopting shotgun sequencing using a high-throughput Illumina MiSeq sequencing platform (Illumina, Inc., USA) at THSTI. Approximately, 100 ng of pure genomic DNA was used for DNA fragmentation and library preparation and pair-end sequencing using Nextera XT DNA Library preparation kit (Illumina, Inc., USA). FastQC and Trimmomatic programs were used to review the quality of raw reads and remove adapter sequences and low quality reads. An average of 4,03,396 clean quality filtered reads were used to generate the draft genome of 10 Lactobacillus isolates belonging to two different species. The average sequencing coverage of the genomes was ~34.63 times. The cleaned pair-end reads were used for genome assembly using Unicycler pipeline (Wick et al., 2017 (link)). After assembly, all the contigs of the genome were annotated by Rapid Annotation Subsystem Technology (RAST) automated annotation pipeline (Aziz et al., 2008 (link)). Annotated proteins were further confirmed by comparing their sequence homology with the reported proteins publicly available in the Protein database (PDB). Around 98% genes predicted to encode proteins were also available in the PDB. Whole genome sequences of all the Lactobacillus strains will be available immediately after acceptance of the article.

Microbial DNA was extracted using the FastDNA^® Spin Kit for Soil (MP Biomedicals, USA) according to the manufacturer’s instructions. All DNA samples were quality checked and the concentration was quantified by NanoDrop 2000 (Thermo Fisher Scientific, USA). Bacterial 16S rRNA gene fragments (V3–V4) were amplified from the extracted DNA using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplification was performed in 20-μL reactions with TransStart Fast Pfu DNA Polymerase (TransGen Biotech, China), 5 μM of each primer and 10 ng of template. The reactions were performed under the following thermal profile: 95 °C for 3 min, followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, and one cycle of 72 °C for 10 min and a 4 °C hold. PCR products were examined by 2% agarose gel electrophoresis and then purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). Amplicons were quantified using the Quantus™ Fluorometer (Promega, USA). Amplicons were subjected to paired-end sequencing on the Illumina MiSeq sequencing platform at Majorbio Bio-Pharm Technology Company (Shanghai, China).