Soil genomic DNA was extracted from the sample using the E.Z.N.A. soil DNA kit (Omega Biotek, GA 30,071, USA). The extracted DNA was purified and quantified by spectrophotometer. (Thermo, MA 02,451, USA). The forward and reverse primers were connected with the universal primer of Illumina Miseq high-throughput sequencing platform, and the PCR products with universal primer sequence at both ends were obtained by the first round PCR reaction using genomic DNA as template, and the PCR products obtained in the first round were purified. The PCR product of the sequence was obtained by connecting the two ends of the sequencing tag sequence with the primer sequence matched with the general primer sequence of the first round of PCR. The purified PCR product was used as the template for the second round of PCR reaction. The amplicon extracted from 2% agarose gel was purified by PCR purification kit (Beckman, Indiana 46,268, USA) and quantified using a Qubit® 2.0 fluorimeter (Invitrogen, CA 92,008, USA). The Illumina Miseq high-throughput sequencing platform was used for sequencing (Shanghai Sangon Biotechnology Co., LTD., Shanghai, China).
Miseq high throughput sequencing platform
Manufactured by Illumina
Sourced in United States, China
The MiSeq is a high-throughput sequencing platform designed for targeted sequencing applications. It utilizes sequencing-by-synthesis technology to generate high-quality sequence data.
Automatically generated - may contain errors
Lab products found in correlation
E z n a soil dna kit, by Omega Bio-Tek (3 mentions)
Superscript 3 one step rt pcr system with platinum taq high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Nextera xt dna preparation kit, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Agilent dna 1000 kit, by Agilent Technologies (2 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Dneasy mericon food kit, by Qiagen (1 mentions)
Qubit 2.0 fluorimeter, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Dna mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Magmax core nucleic acid purification kit, by Thermo Fisher Scientific (1 mentions)
Magmax viral pathogen nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Nextseq platform, by Illumina (1 mentions)
Miseq reagent kit v3 v4, by Illumina (1 mentions)
Superscript 3 one step rt pcr system, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 uorimeter, by Thermo Fisher Scientific (1 mentions)
21 protocols using miseq high throughput sequencing platform
1
Soil Metagenomic DNA Extraction and Sequencing
2
Soil Metagenome Sequencing Protocol
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Soil genomic DNA was extracted from the sample using the E.Z.N.A. soil DNA kit (Omega Biotek, GA 30071, USA). The extracted DNA was puri ed and quanti ed by spectrophotometer. (Thermo, MA 02451, USA). The forward and reverse primers were connected with the universal primer of Illumina Miseq high-throughput sequencing platform, and the PCR products with universal primer sequence at both ends were obtained by the rst round PCR reaction using genomic DNA as template, and the PCR products obtained in the rst round were puri ed. The PCR product of the sequence was obtained by connecting the two ends of the sequencing tag sequence with the primer sequence matched with the general primer sequence of the rst round of PCR. The puri ed PCR product was used as the template for the second round of PCR reaction. The amplicon extracted from 2% agarose gel was puri ed by PCR puri cation kit (Beckman, Indiana 46268, USA) and quanti ed using a Qubit® 2.0 uorimeter (Invitrogen, CA 92008, USA). The Illumina Miseq high-throughput sequencing platform was used for sequencing (Shanghai Sangon Biotechnology Co., LTD., Shanghai, China).
3
Gut Microbiome Profiling via 16S rRNA Sequencing
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The gut microbiota of each patient was isolated, and a QIAamp DNA stool minikit (Qiagen, Hilden, Germany) was used for metagenomic DNA extraction. The quality of the metagenomic DNA was assessed by spectrophotometry. We selected the bacterial V3-V4 region of the 16S rRNA gene for high-throughput sequencing (45 (link)). After PCR amplification, the amplicons were quantified, and all PCR products were subsequently pooled in equimolar ratios to reach a final concentration of 100 nmol/liter each. Next, samples were loaded onto an Illumina MiSeq high-throughput sequencing platform for sequencing (17 (link)). The QIIME (v1.7) (46 (link)) platform was used for the bioinformatics analysis. Representative operational taxonomic units (OTUs) were selected and annotated by the Ribosomal Database Project (RDP) for further microbial structure and taxonomic analyses (47 (link)).
4
Full-length Genome Sequencing of Influenza A/H3N2 Viruses
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Viral RNA was extracted from 200 μl of the infected allantoic fluid of embryonated chicken egg or cell culture supernatant with QIAamp viral RNA mini kit (Qiagen, Inc.). For each virus strain, the RNA was eluted in 50 µl nuclease-free water. Complementary DNA was synthesized by reverse transcription reaction, and gene amplification by PCR was performed, whole genome sequencing of influenza A virus [14 (link)] was performed on the MiSeq high-throughput sequencing platform (Illumina, Inc., San Diego, CA, USA).This resulted in the generation of full-length genome sequences of 1417 influenza A/H3N2 viruses isolated across China since 2015.
5
Microbial Profiling of Saliva Samples
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The methods for gDNA extraction and 16S rRNA gene sequencing were as described in our previous study (22 (link)). Bacterial genomic DNA isolation was performed on all saliva samples using E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.). Then the extraction quality, concentration, and purity of DNA were tested. PCR amplification experiments were performed on the V3-V4 region of the microbial 16S rRNA gene, and the PCR product was recovered, purified, and quantified. Library construction was performed using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing analysis was performed using Illumina Miseq high-throughput sequencing platform (22 (link)). The raw sequence data of this study were stored in the Sequence Read Archive (SRA) database of NCBI. The accession number of the database is PRJNA 784772.
6
Macrogenomic DNA Extraction and Sequencing
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After the Douchi sample was ground evenly, 5 g was added to 45 ml normal saline. After beating for 10 min with a beater and centrifugation at 400 r/min for 5 min, the supernatant was again centrifuged at 10,000 r/min for 10 min. The macrogenomic DNA was extracted from the precipitate by QIAGEN DNeasy Mericon Food Kit, and the DNA samples were stored at −20°C (Wang et al., 2018 ).
The extracted macrogenomic DNA was amplified by PCR using the forward primer SSU0817F (5'‐TTAGCATGGAATAATRRAATAGGA‐3') and the reverse primer SSU1196R (5'‐TTAGCATGGAATARAATAGGAMAULY‐3'). Furthermore, seven nucleotide tags were added to the forward primer (Lin,2019 ). The PCR amplification system was as follows: 0.8 μL forward primer (5 μmol/L), 0.8 μl reverse primer (5 μmol/L), 2 μl dNTPs mix (2.5 mmol/L), 4 μl 5× PCR buffer, 10 ng DNA template, and 0.4 μl DNA polymerase (5 U/μl). The mix was supplemented with ddH2O to 20 μl. The PCR amplification conditions were as follows: 95°C 3 min, 95°C 30 s, 55°C 30 s, 72°C 45 s, 30 cycles; final extension at 72°C for 10 min (Tall & Meyling, 2018 ). The DNA products were sequenced by Illumina MiSeq high‐throughput sequencing platform.
The extracted macrogenomic DNA was amplified by PCR using the forward primer SSU0817F (5'‐TTAGCATGGAATAATRRAATAGGA‐3') and the reverse primer SSU1196R (5'‐TTAGCATGGAATARAATAGGAMAULY‐3'). Furthermore, seven nucleotide tags were added to the forward primer (Lin,
7
Sequencing of H9N2 Avian Influenza Strains
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A total of 62 H9N2 strains including 61 environmental strains isolated in the surveillance efforts and one human isolate mentioned above were sequenced by the next-generation-sequencing method (Supplementary Table S1 ). The total viral RNA of these strains was extracted with the Qiagen RNeasy Mini Kit (Lot. 74,104). The RNA was subjected to reverse transcription and amplification using the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (cat#: 12574035, Invitrogen). The DNA library was prepared using Nextera XT DNA Preparation Kits (cat#FC-131-1,096, Illumina). Whole-genome sequencing was then performed on MiSeq high-throughput sequencing platform (Illumina, Inc., San Diego, CA, United States), and the data were analyzed using CLC Genomics Workbench software.
8
16S rRNA and ITS Sequencing of Root Microbiome
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Roots were shaken to remove the loosely adhering soil. Then, roots, stems and leaves washed with PBS for 3 times, and the organs were re‐dip in the PBS for 20 mins. The scrubbing solution was centrifuged for 10 min at 12 000 g for microbial sediment collection and further microbiome analysis. Then, a QIAamp® DNA Mini kit (Qiagen, Hilden, Germany) was used for metagenomic DNA extraction. The quality of the metagenomic DNA was assessed using spectrophotometry. We selected the bacterial V3‐V4 region of the 16S ribosomal RNA (rRNA) gene as well as the fungus ITS region for high‐throughput sequencing analysis (Dethlefsen and Relman, 2011 ). After PCR amplification, the amplicons were quantified, and all PCR products were then pooled to a final concentration of 100 nm . Next, samples were loaded onto an Illumina MiSeq high‐throughput sequencing platform for sequencing (Shao et al., 2017 ). The QIIME (v1.9; Caporaso et al., 2010 ) platform was used for the bioinformatics analysis. Before bioinformatic analysis, the primers and sequence barcodes were removed using Sickle software. Representative OTUs were selected and annotated using the Ribosomal Database Project (RDP) and Greengenes (version 13.8) to determine the phylogeny and relative abundance of the OTUs (Cole et al., 2007 ).
9
Gut Microbiome and Fungal ITS Analysis
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The gut microbial genomic DNA was extracted from stool samples using a stool DNA extraction kit (QIAamp DNA Stool Mini Kit; cat. no. 51504). The primers of the bacterial 16S rDNA were as follows: The upstream primer was 5′-GTGCCAGCMGCCGCGGTAA-3′, and the downstream primer was 5′-GGACTACHVGGGTWTCTAAT-3′. The V4 region of stool bacterial 16S rDNA and the ITS region of fungi were amplified with a high-fidelity enzyme. The ITS1 region primer is ITS1-5F–ITS2; the ITS2 region primer is: ITS2-3F–ITS2- 4R. The library was constructed using New England Biolabs’ NEB Next® UltraTM DNA Library Prep Kit for Illumina library. Sequencing was completed using a Paired-End (PE) approach on the Illumina MiSeq high-throughput sequencing platform.
10
Fecal Microbiome Profiling by 16S rRNA Sequencing
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Approximately 5 g of the middle section of the feces was collected and immediately frozen and stored at ‒80°C. The samples were transported on dry ice to Shenzhen Micro Health Gene Technology Co., Ltd. for high-throughput sequencing. MoBio's PowerSoil® DNA Isolation Kit was used to extract bacterial DNA from fecal samples. Amplification of the V3 – V4 region of the 16S rRNA gene in DNA was performed by polymerase chain reaction (PCR). Amplified samples were sequenced using the Illumina MiSeq high-throughput sequencing platform.
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