16S 多样性材料与方法
1.1 Sample Collection (此部分请老师根据自己试验内容进行撰写，且后面标黄部分都请
结合具体情况填写， PCR 引物和条件可参考 PCR 质检报告)
1. 2 DNA extraction and PCR amplification
Microbial community genomic DNA was extracted from *** samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R(5'-GGACTACHVGGGTWTCTAAT-3') by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95 ℃ for 3 min, followed by 27 cycles of denaturing at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s and extension at 72 ℃for 45 s, and single extension at 72 ℃ for 10 min, and end at 4 ℃. The PCR mixtures contain 5 × TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL , forward primer (5 μM) 0.8 μL, reverse primer (5 μM) 0.8 μL, TransStart FastPfu DNA Polymerase 0.4 μL, template DNA 10 ng, and finally ddH2O up to 20 μL. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA).（请老师根据您的 PCR 扩增报告进行相应更改）
1.3 Illumina MiSeq sequencing
Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego,USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP******).
1.4 Processing of sequencing data
The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0[1] and merged by FLASH version 1.2.7[2] with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded; (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matching.
Operational taxonomic units (OTUs) with 97% similarity cutoff[3, 4] were clustered using UPARSE version 7.1[3], and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2[5] against the 16S rRNA database (eg. Silva v138) using confidence threshold of 0.7.
References
[1] Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884‐i890. doi:10.1093/bioinformatics/bty560
[2] Magoč T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 2011;27(21):2957‐2963. doi:10.1093/bioinformatics/btr507
[3] Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10(10):996‐998. doi:10.1038/nmeth.2604
[4] Stackebrandt E , Goebel B M . Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology[J]. International Journal of Systematic Bacteriology, 1994, 44(4):846-849. doi:10.1099/00207713-44-4-846
[5] Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73(16):5261‐5267. doi:10.1128/AEM.00062-07
