Approximately 0.2 g of faecal material was used per extraction. DNA was extracted from samples using NucleoSpin® 96 Soil (Macherey-Nagel). Bead beating was done on a Vortex-Genie 2 horizontally for 5 min. A minimum of one negative control was included per batch of samples from the DNA extraction and throughout the laboratory process (including sequencing). A ZymoBIOMICS™ Microbial Community Standard (Zymo Research) was also included in the analysis.
Nucleospin 96 soil
Manufactured by Macherey-Nagel
Sourced in Germany, France
The NucleoSpin® 96 Soil is a high-throughput DNA extraction kit designed for the purification of genomic DNA from soil samples. It utilizes a silica-based membrane technology to efficiently capture and purify DNA, while removing common PCR inhibitors. The kit is suitable for processing multiple samples simultaneously in a 96-well format.
Automatically generated - may contain errors
Lab products found in correlation
Zymobiomics microbial community standard, by Zymo Research (3 mentions)
Miseq reagent kit v3, by Illumina (3 mentions)
Miseq 300pe, by Illumina (2 mentions)
Miseq desktop sequencer, by Illumina (2 mentions)
Miseq sequencer, by Illumina (1 mentions)
Vortex genie 2, by Scientific Industries (1 mentions)
Qubit 2.0 fluorometer, by Agilent Technologies (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
8 protocols using nucleospin 96 soil
1
Microbial Diversity Profiling from Feces
2
Metagenomic Analysis of Gut Microbiome
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DNA was extracted from ∼0.1 g aliquots of the fecal samples using the NucleoSpin 96 Soil (Macherey-Nagel) kit. Fragmented DNA was used for library construction using a NEBNext Ultra Library Prep Kit for Illumina (New England Biolabs). Quantitative real-time PCR (qPCR) was used to determine the concentration of the final library before sequencing. The library was sequenced using 2 × 150-bp paired-end sequencing on the Illumina platform. The metagenomic analysis was performed using the metagenomic species (MGS) concept (17 (link)) and the Clinical Microbiomics human gut MGS database. For taxonomic abundance profiling, we used the Clinical Microbiomics HGMGS version HG4.D.1 set of 2095 metagenomic species (MGS), each represented by a set of genes with highly coherent abundance profiles and base compositions in the 12,170 metagenomes, which includes 481 from infants, 9,428 publicly available metagenomes (18 (link)), and 3,567 publicly available genome assemblies from isolated microbial strains. Individual high-quality non-host reads were mapped to a gene if the mapping quality (MAPQ) was ≥ 20, and the reads aligned with ≥ 95% identity over ≥ 100 base pairs.
3
Fecal DNA Extraction Protocol
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DNA was extracted from ~ 0.1 g aliquots of the fecal samples using the NucleoSpin 96 Soil (Macherey–Nagel, Hoerdt, Germany) kit. Bead beating was done horizontally on a Vortex-Genie 2 at 2700 rpm for 2 × 5 min. The samples were almost intact after only 5 min (standard) bead beating; the stool consistency was dry and hard. Double bead beating improved the DNA concentrations of the samples. Two samples did not have sufficient material for re-extraction and were therefore only subjected to 5 min bead beating. A minimum of one negative control was included per batch of samples from the DNA extraction step and throughout the laboratory process (including sequencing). A ZymoBIOMICS Microbial Community Standard (Zymo Research, Freiburg im Breisgau, Germany) was included in the analysis as a positive (mock) control.
4
Microbiome Profiling of Mouse Cecal Samples
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Mouse cecal samples (12 per group) were shipped to Clinical Microbiomics Lab (Copenhagen, Denmark) for 16S DNA sequencing according to the standard routines performed at the lab. Briefly, DNA of 88 mouse cecal samples were successfully extracted using NucleoSpin® 96 Soil (Macherey-Nagel) with additional bead-beating steps. The V3-V4 region of the 16S rRNA genes were amplified and sequenced on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2 × 300 bp paired-end sequencing. Data obtained from an average sequencing depth of 28,194 read pairs per sample after quality filtering (Illumina MiSeq 300PE) were used for bioinformatics analysis. An adjusted dada2 pipeline was used to process the sequence data into an amplicon sequence variant abundance table (62 (link)). The default taxonomic assignment of the detected ASVs was done using a naïve Bayesian classifier algorithm comparing the ASV sequences to the SILVA reference database (v138.1).
5
Microbiome Analysis of Exacerbation Frequency in COPD
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The microbiological and statistical analyses of plaque samples were performed at Clinical Microbiomics, Copenhagen. DNA was extracted using NucleoSpin® 96 Soil (Macherey-Nagel). Polymerase chain reaction was conducted using the forward primer S-D-Bact-0341-b-S-17 and reverse primer S-D-Bact-0785-A-A-21 with Illumina adapters attached.22 (link) The samples were sequenced in an Illumina MiSeq sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2 × 300 bp paired-end sequencing, and an adjusted dada2 pipeline processed the sequence data into the amplicon sequence variant (ASV) abundance table.23 (link) Taxonomic assignment of the detected ASVs was conducted using a naïve Bayesian classifier algorithm comparing the ASV sequences to the expanded Human Oral Microbiome Database (eHOMD v.15.21).24 (link) Microbiological richness (alpha diversity) was assessed as observed richness (number of ASVs present in a sample) and the Shannon index (ASV composition). Dissimilarity in the taxonomic composition among samples (beta diversity) was calculated as Bray-Curtis dissimilarity.
A proprietary machine learning pipeline based on the R package SIAMCAT25 (link) was used to infer associations between ASVs from the baseline samples and differences in exacerbation frequency and CAT score at the 12-month follow-up visit from those at baseline.
A proprietary machine learning pipeline based on the R package SIAMCAT25 (link) was used to infer associations between ASVs from the baseline samples and differences in exacerbation frequency and CAT score at the 12-month follow-up visit from those at baseline.
6
Longitudinal Gut Microbiome Profiling in T1D
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Stool samples were collected from the INNODIA ND cohort at baseline (within 6 weeks after the diagnosis of type 1 diabetes) and at 3, 6, 12 and 24 months later. In the INNODIA UFM cohort, stool samples were collected at 6, 12, 18, 24 and 36 months after the screening visit (baseline). The samples were collected in OMNIgene-Gut OMR-200 collection tubes (DNA Genotek, Ottawa, ON, Canada), which stabilise DNA at ambient temperature for up to 60 days. Participants were asked to collect the stool samples at home during the week preceding the next study centre visit and to bring the sample with them to the study centre. The samples were frozen at -80°C in the local study centre and shipped frozen to the INNODIA Biobank for storage at -80°C until analysis.
DNA extraction DNA was extracted from 250 μl aliquots of the faecal samples in OMNIgene-Gut OMR-200 collection tubes using the NucleoSpin 96 Soil (Macherey-Nagel, Hoerdt, France) kit. Bead beating was done horizontally on a Vortex-Genie 2 (Scientific Industries, NY, USA) at 2700 rev/min for 5 min. One negative control and one positive control (Zymo-BIOMICS Microbial Community Standard, Zymo Research, CA, USA) were included per batch of samples from the DNA extraction and throughout the laboratory process.
DNA extraction DNA was extracted from 250 μl aliquots of the faecal samples in OMNIgene-Gut OMR-200 collection tubes using the NucleoSpin 96 Soil (Macherey-Nagel, Hoerdt, France) kit. Bead beating was done horizontally on a Vortex-Genie 2 (Scientific Industries, NY, USA) at 2700 rev/min for 5 min. One negative control and one positive control (Zymo-BIOMICS Microbial Community Standard, Zymo Research, CA, USA) were included per batch of samples from the DNA extraction and throughout the laboratory process.
7
Metagenomic DNA Extraction and Library Preparation
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Prior to DNA extraction the rectal swabs were defrosted on ice and centrifuged at 300× g for 10 min, to pellet a large part of the epithelial cells. The supernatant was carefully transferred to a new Eppendorf. DNA was extracted using NucleoSpin® 96 Soil (Macherey-Nagel). Bead beating was done on a Vortex-Genie 2 horizontally for 5 min. The genomic DNA was randomly sheared into fragments of around 350 bp using ultrasonic interruption (Bioruptor Pico, Diagenode SA). The fragmented DNA was applied for library construction using NEBNext Ultra II Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The prepared DNA libraries were evaluated using a Qubit 2.0 fluorometer and Agilent 2100 Bioanalyzer. Quantitative real-time PCR (qPCR) was used to determine the concentration of the final library before sequencing. The library was sequenced using 2 × 150 bp paired-end sequencing on an Illumina platform (Novaseq6000).
8
Microbiome Profiling of Mouse Cecal Samples
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Mouse cecal samples (12 per group) were shipped to Clinical Microbiomics Lab (Copenhagen, Denmark) for 16S DNA sequencing according to the standard routines performed at the lab. Briefly, DNA of 88 mouse cecal samples were successfully extracted using NucleoSpin® 96 Soil (Macherey-Nagel) with additional bead-beating steps. The V3-V4 region of the 16S rRNA genes were amplified and sequenced on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2 × 300 bp paired-end sequencing. Data obtained from an average sequencing depth of 28,194 read pairs per sample after quality filtering (Illumina MiSeq 300PE) were used for bioinformatics analysis. An adjusted dada2 pipeline was used to process the sequence data into an amplicon sequence variant abundance table (62 (link)). The default taxonomic assignment of the detected ASVs was done using a naïve Bayesian classifier algorithm comparing the ASV sequences to the SILVA reference database (v138.1).
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