High-Throughput Method for Preparation of 16S rRNA Gene Sequencing Libraries for Plant Root Microbial Community Profiling

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Sample collection

Soy (Glycine max (L.) Merr., Beijing) was sown in a sandy field at Arid Land Research Center, Tottori University, Japan in July 2018. The plants were grown under two water conditions (drought block well watered and limited in water) in two aftershocks. White mulching sheets (Tyvek, Dupont, US) and sprinkler tubes were installed to control soil conditions. Artificial irrigation from sprinkler tubes was applied for 5 h per day in the well-watered blocks, while no artificial irrigation was used in the limited-water blocks from 14 days after sowing. . Sixty-two days after sowing, the roots of the plants were harvested and washed with tap water. The tips (~2 cm in length) of lateral roots developed from the main roots 0–10 cm from the shoot/root junction were collected and stored at −80 °C until sample preparation. The sampled root tissues were thought to contain endophytes and may also contain bacteria remaining from the rhizoplane. For a simulated bacterial community experiment, we used a soil sample with the ZymoBIOMICS Microbial Community Standard (Zymo Research, Cat. # D6300), where we added 5 μL of the mock microbial community standards into 500 mg of gray soil lowlands. To compare our method with commonly used DNA extraction kits (KIT1/2, details in next section), we used 5 plant root samples and 4 soil samples taken from different plant species (roots of soybeans, rice roots and Andropogoneae sp. roots) and different sites (brown forest floor [36° N, 140° E]plain gray soil [37° N, 140° E; 43° N, 141° E; 33° N, 130° E]paddy field peat soil [43° N, 141° E]).

DNA extraction

The collected root tissues were cooled with liquid nitrogen and immediately ground into a fine powder at 3000 rpm for 15 s using a Multi-Beads Shocker (Yasui Kikai Co. Osaka, Japan, Cat .#MB2200(S)). For each of the tissue samples collected, 500 mg of the powdered sample was transferred to a 1.5 ml tube cooled with liquid nitrogen. One mL of Lysate Binding Buffer (1 M LiCl Sigma-Aldrich, Cat. #L7026-500ML; 100 mM Tris–HCl, Wako, Cat. #318-90225; 1% SDS, Wako, Cat. #313-90275 10 mM EDTA pH 8.0, Wako, Part No. 311-90075, Defoamer A, Sigma-Aldrich, Part No. A5633-25G, 5 mM DTT, Wako, Part No. 048-29224, 11.2 M 3 -Mercapto-1,2-propanediol, Wako, Cat. No. 139-16452;DNase/RNase-free H2O, Thermo Fisher Scientific, Cat. #10977015)36 was added to the sample, which was then homogenized by vortexing, followed by incubation at room temperature (~22°C) for 5 min. The tube was centrifuged at 15,000 rpm for 10 min at room temperature, and the supernatant (LBB lysate) was transferred to a new 1.5 ml tube. DNA extraction was performed using the following two methods: our custom protocol involving isopropanol, acetone, RNase treatment, ethanol precipitation method36 as a manual method of control, and extraction using AMPure XP beads (Beckman Coulter, Cat. #A63881). DNA concentration and absorbance were measured with a spectrophotometer (NanoDrop OneVS Microvolume UV-Vis Spectrophotometer with Wi-Fi, Thermo Fisher Scientific, Cat. #ND-ONEC-W) as well as a fluorimeter (Qubit™ 4 Fluorometer, Thermo Fisher Scientific, Cat. #Q33238). To compare our method with commonly used kits, we used the same amount of soil samples to extract DNA with the DNeasy PowerSoil Pro Kit (denoted KIT1, QIAGEN, Cat. #47014) and the FastDNA SPIN Kit for the soil (denoted KIT2, GEN, Cat. #6560-200) following the manufacturer’s protocols.

Manual DNA extraction method (Control 1, CON1)

The detailed method has been described in our previous publications36.37 and has also been used for soil and plant root microbial community profiling14. Briefly, LBB lysate (200 μL) was added to a 1.5 mL tube and 5 μL of 10 mg/mL proteinase K was added; the mixture is then incubated at 37° C. for 30 min. Then, 200 μL of 100% isopropanol was added to this sample, and this mixture was mixed gently, incubated at room temperature for 5 min and centrifuged at 15,000 rpm for 5 min. The supernatant was discarded to avoid pellet loss and 400 μL of 100% acetone was added to the tube. This was mixed gently, incubated at room temperature for 5 min and centrifuged at 15,000 rpm for 5 min. The supernatant was carefully discarded to avoid pellet loss, and the pellet was dried. After repeated destaining with acetone, we added 100 μL of 10 mM Tris–HCl (pH 7.5) to the tube, incubated at 65°C for 10 min, and then centrifuged at 15,000 rpm for 1 min. The supernatant was transferred to a new 1.5 mL tube and 1 μL of 1 μg/μL RNase A was added and incubated at 37°C for 15 min. The supernatant was added to 10 μL of 3 M ammonium acetate and 250 μL of 100% ethanol, mixed, incubated at room temperature for 5 min and centrifuged. The supernatant was discarded and 400 μL of 80% ethanol was added, mixed, incubated at room temperature for 2 min and centrifuged at 15,000 rpm for 1 min. The supernatant was carefully discarded to avoid pellet loss, and the pellet was dried. DNA was eluted in 50 μL of 10 mM Tris-HCl (pH 7.5).

AMPure XP Bead Method for DNA Extraction (High Throughput Option 1, HTO1)

Fifty μL of LBB lysate was placed in 1.5 mL tubes and an equal amount of AMPure XP beads added, followed by incubation at room temperature for 5 minutes after vortexing. The mixture was placed on a magnetic station for 5 minutes and the supernatant was discarded. The magnetic beads were washed twice with 200 μL of 80% ethanol. Finally, the DNA was eluted with 20 μL of 10 mM Tris-HCl (pH 7.5).

16S rRNA gene sequencing

Library preparation using a two-step PCR amplification protocol was reported in our previous publication.14. In this study, we compared two purification methods: purification based on magnetic beads and exonuclease after the first step of PCR. Briefly, the V4 region of the bacterial 16S rRNA gene was amplified with primers 515f and 806rB (forward primer: 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG- [3–6-mer Ns]—GTG YCA GCM GCC GCG GTA A-3′; reverse primer: 5′- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G [3–6-mer Ns]—GGA CTA CNV GGG TOP CTA AT-3′)21.38 Each sample (1 μL DNA diluted ten times) was amplified in a 10 μL reaction volume containing 0.2 U KOD FX Neo DNA polymerase (TOYOBO Co., Ltd., Osaka, Japan), 2 × PCR buffer (TOYOBO), 0.4 mM dNTPs (TOYOBO), 0.2 μM forward and reverse primers, and 1 μM blocking primers (mPNA and pPNA, PNA BIO, Inc., Newbury Park, CA). PCR was performed using the following specifications: 94°C for 2 min followed by 35 cycles at 98°C for 10 s, 78°C for 10 s, 55°C for 30 s, 68°C for 50 s, and a final extension at 68°C for 5 min (ramp rate = 1°C/s). The PCR products were then purified by two separate methods (see 3–1 and 3–2). The second PCR was carried out with the following primers: forward primer: 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC AC-[8-mer index]—TCG TCG GCA GCG TC -3′, and reverse primer: 5′- CAA GCA GAA GAC GGC ATA CGA GAT—[8-mer index]—GTC TCG TGG GCT CGG-3′39. Each sample (0.8 μL of purified product from the first PCR) was amplified in a 10 μL reaction volume containing 0.2 U KOD FX Neo DNA polymerase (TOYOBO), 2 × PCR buffer (TOYOBO), 0, 4 mM dNTPs (TOYOBO), 0.3 μM forward and reverse primers and 1 μM blocking primers (mPNA and pPNA). PCR was performed as follows: 94°C for 2 min, followed by 8 cycles of 98°C for 10 s, 78°C for 10 s, 55°C for 30 s, 68°C for 50 s and an extension final at 68°C for 5 min (ramp rate = 1°C/s). After amplification, PCR products for each sample were cleaned and size-selected using AMPure XP beads and washed twice with 80% ethanol. Libraries were eluted from the pellet with 10 µL of 10 mM Tris–HCl pH 7.5, quantitated with a microplate photometer (Infinite 200 PRO M Nano+, TECAN Japan Co., Ltd.), and grouped into a single library in equal molar amounts. The pooled library was sequenced on an Illumina MiSeq platform using a MiSeq v3 reagent kit (600 cycles) and a MiSeq Nano v2 reagent kit (500 cycles) (Illumina, CA, USA) .

AMPure XP Bead Method for PCR Purification (Control 2, CON2)

A solution containing AMPure XP beads (10 μL) was added to 10 μL of product from the first PCR, and the mixture was incubated at room temperature for 5 min after mixing with a vortex. The mixture was then placed on a magnetic station for 5 min, and the supernatant was then discarded. The magnetic beads were washed twice with 200 μL of 80% ethanol. The purified sample was eluted from the beads by incubation with 10 μL of 10 mM Tris-HCl (pH 7.5).

Exonuclease method for PCR purification (High Throughput Option 2, HTO2)

Two μL of ExoSAP-IT Express (Thermo Fisher Scientific, Cat #75001.1.EA) was added to 5 μL of the product obtained from the first PCR, and the mixture was incubated at 37°C for 4 min, then at 80°C for 1 min.

Bioinformatics

Bioinformatics and statistical analyzes were performed using the Quantitative Insights Into Microbial Ecology 2 program (QIIME 2, ver. 2020.6.0, https://qiime2.org/) installed via docker40. Matched raw FASTQ files were imported into the QIIME2 program and demuxed using a native plugin. Subsequently, the Cutadapt plugin was treated primer trimmed. The Divisive Amplicon Denoising Algorithm 2 (DADA2) plugin in QIIME2 was used for quality filtering. Demuxed FASTQ file has been cut, denoised, chimera removed and data merged41. We applied the setting with a truncation length of 220 for forward and reverse playback. Taxonomic groups were assigned an identity with the Naive Bayes q2-feature-classifier formed using the 515F/806R region from 99% of the operational taxonomic units (OTUs) of the SILVA 138 rRNA database42.43. Contaminating sequences of archaea, eukaryotes, mitochondria, and chloroplasts were filtered from the resulting feature table. After taxonomic assignment of amplicon sequence variants (ASVs), the remaining representative sequences were aligned with MAFFT and used for phylogenetic reconstruction in IQ-TREE multicore version 2.0.344. The sample depth parameter was set to 1181 (HTO1/2 vs. CON1/2, Supplemental Dataset 1) and 2988 (HTO1_HTO2 vs. KIT1/2_CON2, Supplemental Dataset 2), which were chosen based on function of the number of sequences in the sample containing the smallest number of sequences (Supplementary Fig. S1). Finally, diversity indices such as Shannon diversity, Faith phylogenetic diversity, Bray-Curtis distance and weighted UniFrac distance were calculated using the QIIME2 diversity plugin. The resulting data was exported as a BIOM table and imported into the LDA Effect Size (LEfSe) algorithm to determine biomarker differences45. The LEfSe was performed with the following parameters: Kruskal-Wallis non-parametric factorial test, Wilcoxon pairwise test (P 2.0. Rank-Abundance Dominance (RAD) analysis was performed using the R package RADanalysisworm. 0.5.546. These RAD curves display the logarithmic abundances of species in rank order using minimum richness (R= 40) to normalized data.

Ethical statements

All experiments using plant materials complied with local and national regulations.

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