The total genomic DNA was extracted from a clean colon stool sample. The V3-V4 region of the 16S rRNA gene was amplified with specific primers. Samples were sequenced on the IonS5TMXL platform provided by Novogene (Beijing, China). Using a single-end sequencing method, a small fragment library was constructed for single-end sequencing. Through Reads shear filtering, Operational Taxonomic Units (OTUs) clustering, analysis of the annotation and the abundance of species, alpha diversity analysis, and beta diversity analysis (Beta Diversity), we chose the t-test, MetaStat, and LEfSe statistical analysis methods, such as species composition and community structure of the sample difference significance test.
Ions5tmxl platform
Manufactured by Novogene
Sourced in China
The IonS5TMXL platform is a DNA sequencing system designed for high-throughput genome analysis. It utilizes semiconductor-based sequencing technology to generate DNA sequence data efficiently and accurately.
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Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Ion plus fragment library kit 48 rxns, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Easypure stool genomic dna kit, by Transgene (1 mentions)
Genejettm gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Mobio powersoil dna isolation kit, by Qiagen (1 mentions)
E z n a stool dna kit, by Omega Bio-Tek (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Eznatm soil dna kit, by Omega Bio-Tek (1 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Pacbio sequel system, by Novogene (1 mentions)
9 protocols using ions5tmxl platform
1
Stool Microbiome Analysis by 16S rRNA Sequencing
2
Gut Microbiome Profiling via 16S rRNA Sequencing
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Genomic DNA was extracted from cecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The 16S rRNA gene V3-V4 variable region was PCR-amplified with the universal primer set 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). The PCR products were pooled and purified, and sequencing libraries were generated using the Ion Plus Fragment Library Kit (Thermo Scientific, Waltham, MA, USA). The library was then sequenced on an IonS5TM XL platform at Novogene, Beijing, China. All the raw sequencing data were collected into the NCBI Sequence Read Archive database with accession numbers SRR10714265-SRR10714300.
Uparse V7.0.1001 was used to perform operational taxonomic units (OTUs) clustering of sequences with ≥97% similarity (19 (link)). Representative sequences for each OTU were screened for further annotation. The Chao1 and abundance-based coverage estimator (ACE) indices were calculated with QIIME V1.9.1 (20 (link)) and displayed with R software V2.15.3. Hierarchically clustered heat map and principal component analysis (PCA) based on weighted UniFrac distance matrix were generated by R software V2.15.3. The linear discriminant analysis (LDA) effect size (LEfSe) analysis was employed to identify the significant differences between groups (LDA > 2) by LEfSe software.
Uparse V7.0.1001 was used to perform operational taxonomic units (OTUs) clustering of sequences with ≥97% similarity (19 (link)). Representative sequences for each OTU were screened for further annotation. The Chao1 and abundance-based coverage estimator (ACE) indices were calculated with QIIME V1.9.1 (20 (link)) and displayed with R software V2.15.3. Hierarchically clustered heat map and principal component analysis (PCA) based on weighted UniFrac distance matrix were generated by R software V2.15.3. The linear discriminant analysis (LDA) effect size (LEfSe) analysis was employed to identify the significant differences between groups (LDA > 2) by LEfSe software.
3
Gut Microbiome DNA Extraction and Sequencing
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An EasyPure Stool Genomic DNA Kit (EE301-01; TransGen Biotech, Beijing, China) was used to isolate DNA from 20 g of frozen samples, following the manufacturer’s guidelines. The purity and concentration of the extracted DNA was measured using a Nanodrop 2000 instrument (Thermo, Dreieich, Germany). The 16S rRNA V3–V4 regions were amplified using the universal eubacterial primers (341F: 5-CCTAYGGGRBGCASCAG-3, 806R: 5-GGACTACNNGGGTATCTAAT-3). The amplicons were then purified using a TruSeq DNA PCR Free Preparation Kit (QIAGEN, Dusseldorf, Germany). The amplicons were sequenced on the IonS5TMXL platform (Novogene, Sacramento, CA, USA), which generated 600-bp single-ended reads.
4
Soil Bacterial Community Profiling
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All plant tissues (i.e., roots, stems, leaves and seeds) were ground to a powder in liquid nitrogen prior to DNA extraction. DNA extraction was performed using the MoBio PowerSoil DNA Isolation kit (QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. DNA concentration and purity were monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/µL using sterile water. To access the bacterial communities, the V4 region of the 16S rRNA gene was amplified using the specific primer (V4: 515F-806R) with the barcode. All PCR reactions were carried out in 30 µL reactions with 15 µL Phusion® High-Fidelity PCR Master Mix (Biolabs, New England, Ipswich, MA, USA); 0.2 µM of forward and reverse primers, and about 10 ng of template DNA. PCR products were purified with the GeneJETTM Gel Extraction Kit (Thermo Scientific, Shanghai, China). Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific, Shanghai, China) following the manufacturer’s recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific, Shanghai, China). The library was sequenced on an Ion S5TM XL platform (Novogene, Beijing, China), and 400 bp/600 bp single-end reads were generated.
5
Microbial Characterization of Intestinal Community
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The intestinal community of the micro-organisms was performed according to Suo (Suo et al., 2017) (link) with slight changes. The total DNA of microbes in the intestine was extracted directly with the E.Z.N.A. Stool DNA Kit (Omega Bio-tek, Inc., GA, USA) (Xin et al., 2015) (link). The amplification and sequencing of the hypervariable region (V4 + V5) of the bacterial 16S DNA gene was performed for the sequencing analysis and species identification. High-throughput sequencing was performed on the IonS5TMXL platform (Novogene, China). The sequencing reads were assigned to each sample according to the individual unique barcode. The sequences were analyzed with the QIIME software package (Quantitative Insights Into Microbial Ecology) and the UPARSE pipeline (Caporaso et al., 2011) (link). The reads were first filtered and clustered into operational taxonomic units (OTUs) at an identity threshold of 97%. The alpha and beta diversity analyses were then performed as described by Amoah et al. (2019b) (link).
6
16S rRNA Gene Sequencing of Cecal Microbiome
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Almost 0.5–1.0 g of homogenized cecal chyme of each chick was used. Total genomic DNA was extracted using the EZNATM Soil DNA kit (D5625-02, Omega Bio-Tek Inc., Norcross, GA, United States) and stored at −20°C. The V4 region of bacterial 16S rRNA was amplified by PCR using the primer pair 515F/806R (Bergmann et al., 2011 (link); Gao et al., 2017 (link)). The amplified products containing main fragments of 400–450 bp were extracted and chosen for further analysis (Caporaso et al., 2011 (link); Gao et al., 2017 (link)). PCR products were purified using the GeneJET Gel Extraction Kit (Thermo Scientific, Waltham, MA, United States). After Qubit quantitative and library detection, the individually barcoded 16S rDNA amplicons from each sample were pooled and paired-end sequenced on the IonS5TM XL platform at Novogene Bioinformatics Technology Co., Ltd (Beijing, China), and 250-bp paired-end raw reads were generated.
7
Profiling Microbial Diversity Using 16S rRNA Sequencing
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Microbial diversity was determined by amplifying and sequencing the hypervariable region V3-V4 of the bacterial 16S rRNA gene, using primers containing barcodes and PCR conditions as previously reported (Polka et al., 2015 (link); Li et al., 2019 (link)). All PCR amplification reaction mixtures were examined using 2.0% agarose gel electrophoresis with a loading buffer (containing SYRB green) and mixed in equidense ratios and purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific). HTS was conducted on the Illumina MiSeq (Illumina, United States) according to the manufacturer’s specifications. Sequencing libraries were formed using an Ion Plus Fragment Library Kit 48 rxns (Thermo Fisher Scientific), following the manufacturer’s protocol. The library quality was assessed by using a Qubit@ 2.0 fluorometer (Thermo Fisher Scientific). The library was sequenced on an Ion S5TM XL platform and 400 bp/600 bp single-end reads were generated (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). Raw data from a next-generation sequencing platform were submitted to the Sequence Read Archive of National Center for Biotechnology Information (NCBI), under BioProject ID PRJNA746727 .
8
Microbial Community Analysis of Batch Experiments
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To confirm the dominant microorganisms after batch experiments, microbial samples were obtained from each reactor for sequencing. DNA extractions, amplification and analysis were carried out by Novogene Co. Ltd. (Beijing, China). For polymerase chain reaction (PCR) amplification, a pair of universal 16S rRNA gene primers PS5 (341b4F-806R) was used for high-throughput sequencing [32 (link)]. The details of the microbial analysis were presented in a previous study [33 (link), 34 (link)]. Briefly, DNA was extracted and amplified using the 16S V4 region primers (515F and 806R). Then amplicons were sequenced using the IonS5TMXL platform at Novogene Co. Ltd. (Beijing, China). Filtered sequences were classified as operational taxonomic unit (OTU) with 97% identity using the Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/ ) [35 (link)]. Taxonomy was assigned to OTUs against SSUrRNA database of the SILVA (http://www.arb-silva.de/ ) [36 (link)]. The OTU table was rarefied to 63,933 sequences per sample in QIIME. Further data analysis was performed based on OTUs.
The microbial α-diversity was assessed using three metrics, including the Shannon index [37 (link)], the Observed_species, the Chao1 index [38 (link)], and PD whole tree.
The microbial α-diversity was assessed using three metrics, including the Shannon index [37 (link)], the Observed_species, the Chao1 index [38 (link)], and PD whole tree.
9
Bacterial and Archaeal Community Profiling
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To accurately analyze the bacterial community, the full-length fragment of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) for Single-molecule real-time sequencing (SMRT) using a primer pair (F: 5′-AGAGTTTGATCCTGGCTCAG-3′; R: 5′-GNTACCTTGTTACGACTT-3′). The PCR program was as follows: 95 °C for 2 min; 35 cycles of 95 °C for 30 s, 60 °C for 45 s and 72 °C for 90 s, with a final extension of 72 °C for 10 min (Hou et al., 2015). The barcoded PCR products were resolved by 2% agarose gel electrophoresis, and were purified with a Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA). The SMRT Bell libraries were finally sequenced using the Pacific Biosciences (PacBio) Sequel system (Novogene Co., Ltd., Beijing, China).
To better understand the archaeal community, the V7–V8 region of the 16S rRNA gene was amplified using a primer pair (1106F: 5′-TTWAGTCAGGCAACGAGC-3′ and 1378R: 5′-TGTGCAAGGAGCAGGGAC-3′). After the barcoded PCR products were purified, the library for each sample was constructed and was subjected to single-end sequencing on the IonS5TMXL platform (Novogene).
All raw amplicon sequence data in this study are available at the National Center for Biotechnology Information (NCBI) SRA Science Research Associates (SRA) database under accession: PRJNA593344.
To better understand the archaeal community, the V7–V8 region of the 16S rRNA gene was amplified using a primer pair (1106F: 5′-TTWAGTCAGGCAACGAGC-3′ and 1378R: 5′-TGTGCAAGGAGCAGGGAC-3′). After the barcoded PCR products were purified, the library for each sample was constructed and was subjected to single-end sequencing on the IonS5TMXL platform (Novogene).
All raw amplicon sequence data in this study are available at the National Center for Biotechnology Information (NCBI) SRA Science Research Associates (SRA) database under accession: PRJNA593344.
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