Total DNA from cecal content samples, collected from 20 animals from each Bacillus-fed and control group, were extracted employing the Lysis and Purity kit (Shoreline Biome, Farmington, CT, United States) following manufacturer’s protocol. The resulting DNA was used as template for library preparation using Shoreline Biome’s V4 16S DNA Purification and Library Prep Kit (Shoreline Biome, Farmington, CT, United States). Briefly, PCR amplification of the V4 region of the 16S rRNA gene was performed using the extracted DNA and the primers 515F (5′GTGGCCAGCMGCCGCGGTAA) and 806R (5′-GGACTACHVHHHTWTCTAAT). The resulting amplicons were then sequenced using 2 bp × 150 bp paired-end kits on the Illumina iSeq platform. To increase diversity, PhiX 50 pM was added to a final concentration of 5% into the amplicon library.
Iseq platform
Manufactured by Illumina
Sourced in United States
The ISeq platform is a benchtop next-generation sequencing (NGS) system designed to provide accurate and efficient DNA and RNA sequencing. It is capable of sequencing a wide range of sample types, including genomic DNA, transcriptomes, and targeted panels. The ISeq platform utilizes a proprietary sequencing-by-synthesis technology to generate high-quality sequencing data.
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Lab products found in correlation
Nextera xt library prep kit, by Illumina (2 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Isopropanol, by Carl Roth (1 mentions)
Powerfecal pro dna kit, by Qiagen (1 mentions)
Cfx system, by Bio-Rad (1 mentions)
Qiaseq fx dna library kit, by Qiagen (1 mentions)
Nucleospin plant 2, by Macherey-Nagel (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Superscript 3 one step rt pcr system with platinum taq, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Qiaseq library quant assay kit, by Qiagen (1 mentions)
Qiaseq fx dna library udi kit, by Qiagen (1 mentions)
9 protocols using iseq platform
1
16S rRNA Sequencing of Cecal Samples
2
Influenza Viral RNA Extraction and NGS Analysis
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Viral RNA was extracted with the ReliaPrep™ Cell Mini Kit (Promega, Walldorf, Germany), using a protocol adapted for extraction of RNA from seasonal influenza viruses. First, 200 µL diluted specimen, 50 µL water and 250 µL freshly prepared buffer (BL+TG lysis buffer) were mixed by vortexing. Next, 85 µL of isopropanol (100%, Carl Roth, Karlsruhe, Germany) were added and mixed by vortexing. All subsequent steps (including DNase digestion) were performed according to manufacturer’s instructions. Then, multisegment reverse transcription-PCR was carried out for influenza A and influenza B viruses as described elsewhere.17 (link),18 (link) NGS was performed using 1 ng of purified One-Step RT-PCR product, Nextera XT DNA Library Preparation Kit and subsequently the ISeq platform (Illumina, San Diego, USA). Trimming, reference mapping and generation of consensus sequences were done with Geneious software (11.1.5) and genome sequences (minority variants ≥10%) were deposited in GISAID (www.GISAID.org Supplementary Table 1
NGS data were analyzed for molecular resistance markers in NA, M2 and PA by FluSurver enabled by data from GISAID (https://flusurver.bii.a-star.edu.sg/
NGS data were analyzed for molecular resistance markers in NA, M2 and PA by FluSurver enabled by data from GISAID (
3
Gut Microbiome Analysis of Mice
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Bacterial DNA was isolated from feces using the PowerFecal Pro DNA Kit (QIAGEN). DNA from two mice was combined in equivalent proportions (n = 3–4/group) for 16S rRNA sequencing. The samples were purified and sequenced using the iSeq platform (Illumina, San Diego, CA, USA). Trimmomatic (ver. 0.39) was used to clean the sequences, and then the data were organized into operational taxonomic units and aligned with QIIME2 (version 1.9.5) (accessed on 15 December 2022) [30 (link)]. In addition, α- and β-diversity analyses were conducted using QIIME2. Quantitative PCR was used to validate the results of 16S rRNA sequencing and to quantify the abundance of 10 well-known probiotics in fecal bacteria (CFX System, Bio-Rad). Bacterial DNA was amplified using the specific bacterial primers listed in Supplemental Table S2 (Macrogen). The ratio of total bacteria was used to calculate the relative abundance (F341/R518) (n = 10 per group) [31 (link)].
4
SARS-CoV-2 Genome Sequencing Protocol
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Whole-genome amplification of CoV2 was carried out using a modified version of the ARTIC Network’s protocol for CoV2 genome sequencing (Itokawa et al., 2020 (link)). A next-generation sequencing (NGS) library was constructed using the QIAseq FX DNA Library kit (QIAGEN), and sequenced using an iSeq platform (Illumina). All short-read sequences were mapped to the CoV2 Wuhan-Hu-1 reference genome sequence (GenBank: MN908947.3) using the bwa mem algorithm (version 0.7.13-r1126) (Li and Durbin, 2009 (link)); next, variant allele frequency analysis was conducted using VarScan version 2.4.3 (Koboldt et al., 2012 (link)).
5
Genomic DNA Extraction and VRN1 Gene Sequencing
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Genomic DNA for sequencing was extracted from leaves of two-week-old plants using NucleoSpin Plant II (MACHEREY-NAGEL, Germany) according to the manufacturer’s instructions.
Sequences of VRN1 genes were obtained by sequencing overlapping PCR products using several sequencing protocols as described in (Strejčková et al., 2021 (link)). Briefly, short PCR products (< 1200 bp) were sequenced by the Sanger method, while long PCR products (> 2700 bp) were sequenced with the Illumina iSeq platform.
Sequences of VRN1 genes were obtained by sequencing overlapping PCR products using several sequencing protocols as described in (Strejčková et al., 2021 (link)). Briefly, short PCR products (< 1200 bp) were sequenced by the Sanger method, while long PCR products (> 2700 bp) were sequenced with the Illumina iSeq platform.
6
Gut Microbiome Profiling via 16S rRNA
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Genomic DNA was extracted using PowerFecal Pro DNA kit. DNA was evaluated by 16S rRNA sequencing, and the purity of the samples was measured using an iSeq platform (Illumina, San Diego, CA, USA). Identity of the gut microbiota was quantified at the absorbance ratios at 260/280 nm and amplified using qPCR (CFX Connect Real-Time PCR Detection System, Bio-Rad (Hercules, CA, USA)). PCR conditions were 95 °C for 5 min and 40 cycles at 95 °C for 5 s at the annealing temperature. The primer specificity was verified as follows: 5 min at 65 °C, 5 min at 95 °C, and 0.5 °C increments [34 (link)].
7
Whole-Genome Sequencing of SARS-CoV-2
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The selected swab samples of the patients were then sent to the Chinese CDC for further whole-genome sequencing. Libraries were prepared using a Nextera XT library prep kit (Illumina, San Diego, CA, USA), and the resulting DNA libraries were sequenced on either a MiSeq or an iSeq platform (Illumina) using a 300-cycle reagent kit. Mapped assemblies were generated using the SARS-CoV-2 genome (accession number NC_045512 ) as a reference. Variant calling, genome alignment, and sequence illustrations were generated with CLCBio software. Whole-genome sequence alignment was conducted using the Muscle tool in MEGA (version 7.0). A neighbor-joining phylogenetic tree was constructed using MEGA (version 7.0), and the Kimura 2-parameter model with 1,000 bootstrap replicates was used. Genomic lineage designation was performed using the PANGO lineage typing method (https://cov-lineages.org/ ).
8
Whole-Genome Sequencing of SARS-CoV-2
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The selected swab samples of the patients were then sent to the Chinese CDC for further whole-genome sequencing. Libraries were prepared using a Nextera XT library prep kit (Illumina, San Diego, CA, USA), and the resulting DNA libraries were sequenced on either a MiSeq or an iSeq platform (Illumina) using a 300-cycle reagent kit. Mapped assemblies were generated using the SARS-CoV-2 genome (accession number NC_045512 ) as a reference. Variant calling, genome alignment, and sequence illustrations were generated with CLCBio software. Whole-genome sequence alignment was conducted using the Muscle tool in MEGA (version 7.0). A neighbor-joining phylogenetic tree was constructed using MEGA (version 7.0), and the Kimura 2-parameter model with 1,000 bootstrap replicates was used. Genomic lineage designation was performed using the PANGO lineage typing method (https://cov-lineages.org/ ).
9
Full-genome Sequencing of Influenza Virus Samples
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Full-genome sequencing of selected samples from the animal trials was conducted utilizing universal IAV amplification (SuperScript™ III One-Step RT-PCR System with Platinum Taq, Invitrogen, Waltham, MA, USA) followed by sequencing on an Illumina iSeq platform. After amplification and a succeeding clean-up of the PCR products with magnetic AMPure XP Beads (Beckman-Coulter, Brea, CA, USA), library preparation took place with the QIAseq FX DNA Library UDI Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantification of the prepared libraries was conducted with the QIAseq Library Quant Assay Kit (Qiagen, Hilden, Germany) prior to final pooling. An iterative map-to-reference approach of the sequencing data allowed full-genome production of the samples and further frequency analysis of adaptive mutations in Geneious Prime V.2021.0.1 (Biomatters Ltd., Auckland, New Zealand).
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