Journal: Ecology and Evolution

Article Title: A practical introduction to microbial molecular ecology through the use of isolation chips

doi: 10.1002/ece3.4748

Figure Lengend Snippet: Experimental design of molecular microbial ecology group project. The 18‐week project was divided into three parts: (1) field work, (2) lab work, (3) data analysis and reporting. During fieldwork, students were provided with iChips that they loaded with soil dilutions (1a) and then buried in dedicated locations for 2 weeks (1b). Initial lab work included preparation of soil extract agar plates, plating soil dilutions, and incubated iChip wells (2a) and overlaying isolates from soil and iChips with ESKAPE indicator species (2b). Molecular work included DNA extraction directly from soil samples, and from bacterial colonies recovered from soil dilution plating and isolation chips (iChips) (2c), PCR amplification of 16S rRNA genes and electrophoretic evaluation of PCR products (2d), followed by high throughput amplicon sequencing using the MiSeq platform (2e). Outcomes of the project were assessed through data analysis using QIIME (3a) and the production of a written report (3b)

Article Snippet: Illumina libraries were prepared using a Nextera XT kit, following the manufacturer's recommendations for 16S rRNA gene PCR amplicon barcoding (using 2× NEBNext High‐Fidelity PCR master mix), clean up and pooling.

Techniques: Incubation, DNA Extraction, Isolation, Amplification, High Throughput Screening Assay, Sequencing

Journal: Ecology and Evolution

Article Title: A practical introduction to microbial molecular ecology through the use of isolation chips

doi: 10.1002/ece3.4748

Figure Lengend Snippet: Taxonomic phylum distribution of bacterial communities in soil samples and colonies recovered using spread and iChip isolation techniques from Hagg Farm (HF) and Three Hagges Wood Meadow (THW) sites using 16S rRNA gene amplicon sequencing. The spread plate sample marked with * showed more similarity to the iChip samples based on phylum distribution and PCoA (Figure c). The soil sample marked with # appears to have been sequenced twice

Article Snippet: Illumina libraries were prepared using a Nextera XT kit, following the manufacturer's recommendations for 16S rRNA gene PCR amplicon barcoding (using 2× NEBNext High‐Fidelity PCR master mix), clean up and pooling.

Techniques: Isolation, Amplification, Sequencing

Journal: Ecology and Evolution

Article Title: A practical introduction to microbial molecular ecology through the use of isolation chips

doi: 10.1002/ece3.4748

Figure Lengend Snippet: A heatmap of bacterial genera in soil, spread and iChip retrieved microbial communities based on 16S rRNA amplicon sequencing. Columns with similar annotations were collapsed by calculating the mean for each group. Rows depict identified operational taxonomic units (OTUs) with a summed relative abundance >0.1%. Row names represent the lowest taxonomic rank for a given OTU: g—genus, f—family, o—order, c—class. Rows were centered by subtracting the row means (omitting NAs) of OTUs from their corresponding row; scaling was performed by dividing the (centered) row of OTUs by their standard deviations. The relative abundance of an OTU to which unit variance scaling was applied, in soil, spread and iChip recovered microbial communities ranges from −2 to 2 as shown in the lower heatmap key. Rows were clustered using Euclidean distance and average linkage. Columns were clustered using correlation distance and average linkage. The heatmap was constructed using R pheatmap package (Metsalu & Vilo, ).

Article Snippet: Illumina libraries were prepared using a Nextera XT kit, following the manufacturer's recommendations for 16S rRNA gene PCR amplicon barcoding (using 2× NEBNext High‐Fidelity PCR master mix), clean up and pooling.

Techniques: Amplification, Sequencing, Construct

Journal: Ecology and Evolution

Article Title: A practical introduction to microbial molecular ecology through the use of isolation chips

doi: 10.1002/ece3.4748

Figure Lengend Snippet: Phylogeny of species based on SSuMMo analysis of 60,000 16S rRNA gene sequences sampled from the collected datasets. Only organisms that were at least 0.2% of the analysed reads are included in the trees. Species previously annotated as “uncultured” are indicated with an asterisk (*). Bar heights indicate the relative abundance of reads within each sample. (a) Hagg Farm (HF) samples, (b) Three Hagges Wood Meadow (THW) samples. Species names are provided in Supporting Information Table

Article Snippet: Illumina libraries were prepared using a Nextera XT kit, following the manufacturer's recommendations for 16S rRNA gene PCR amplicon barcoding (using 2× NEBNext High‐Fidelity PCR master mix), clean up and pooling.

Techniques:

Journal: Genes

Article Title: Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

doi: 10.3390/genes11091105

Figure Lengend Snippet: Nose swab samples of individuals and negative controls that were sequenced using and Illumina and nanopore 16S rRNA gene sequencing technologies. (a) = a maximum of 5000 raw Illumina sequence reads were analyzed for the classification of genera. (b) = samples with read numbers below the 500 read cut-off. NA = not applicable.

Article Snippet: 16S rRNA gene sequence libraries were prepared with the 16S Rapid Amplicon Barcoding Kit (Oxford Nanopore Technologies—ONT, Oxford, UK, SQK-RAB201) according to the standard procedures described by ONT.

Techniques: Sequencing, Infection, Control

Journal: Genes

Article Title: Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

doi: 10.3390/genes11091105

Figure Lengend Snippet: Nasal microbiota profiles generated using nanopore and Illumina 16S rRNA gene sequencing. DNA was isolated from 57 nose swab samples, and 16S rRNA gene sequencing was performed using both Illumina ( a ) and nanopore ( b ) technologies. Each bar in the graph represents a nasal microbiota profile from a single individual. The dashed lines in ( b ) represent genera that, by default, were reported as unclassified at genus level in the EPI2ME report but were identified when next to reads with a top three blast hit with one genera (num_genus_taxid is 1); reads with a top three blast hit with two genera (num_genus_taxid is 2) were also included. A phylogenetic tree was generated by Pearson/UPGMA clustering of bacterial genera in microbiota profiles, as determined using Illumina sequencing. To compare between the two techniques, the sample order of the samples that were sequenced with the Oxford Nanopore platform was matched to the sample order of the samples that were sequenced with the Illumina platform, and the percentage of agreement was calculated for each nose swab sample ( c ). The horizontal black line in ( c ) indicates the mean percentage of agreement.

Article Snippet: 16S rRNA gene sequence libraries were prepared with the 16S Rapid Amplicon Barcoding Kit (Oxford Nanopore Technologies—ONT, Oxford, UK, SQK-RAB201) according to the standard procedures described by ONT.

Techniques: Generated, Sequencing, Isolation, Illumina Sequencing

Journal: Genes

Article Title: Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

doi: 10.3390/genes11091105

Figure Lengend Snippet: Agarose gel with 16S rRNA gene amplicons. Total DNA was isolated from pure bacterial cultures in a similar manner as the isolation of DNA from the nasal swab samples; the DNA concentration was determined by picogreen and a PCR was performed as described for nanopore sequencing using equal amounts of template DNA, with the exception that 30 PCR cycli instead of 25 cycli were used.

Article Snippet: 16S rRNA gene sequence libraries were prepared with the 16S Rapid Amplicon Barcoding Kit (Oxford Nanopore Technologies—ONT, Oxford, UK, SQK-RAB201) according to the standard procedures described by ONT.

Techniques: Agarose Gel Electrophoresis, Isolation, Concentration Assay, Nanopore Sequencing

Journal: Genes

Article Title: Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

doi: 10.3390/genes11091105

Figure Lengend Snippet: Genus and species level identification on pure culture species. Pure cultures of bacterial ATCC strains were sequenced using an R9.2 or R9.4 nanopore flowcell and Albacore or Guppy basecalling. Taxonomic assignment was performed at genus ( a ) and species ( b ) level using the EPI2ME 16S pipeline and the following thresholds: read length ≥1400 bp ≤ 1700 bp, num_genus_taxid is 1 or lca is 0 and accuracy ≥80%, QC ≥ 7 when albacore basecalling was used, or accuracy ≥85%, QC score ≥9 when Guppy basecalling was used. Similar criteria and the highest scoring BLAST identification (top rank) was used for species level identification. A is Albacore; G is Guppy basecalling.

Article Snippet: 16S rRNA gene sequence libraries were prepared with the 16S Rapid Amplicon Barcoding Kit (Oxford Nanopore Technologies—ONT, Oxford, UK, SQK-RAB201) according to the standard procedures described by ONT.

Techniques:

Journal: Scientific Reports

Article Title: Oceanographic setting influences the prokaryotic community and metabolome in deep-sea sponges

doi: 10.1038/s41598-022-07292-3

Figure Lengend Snippet: Prokaryotic community composition based on relative abundance of 16S rRNA gene sequences, aggregated at the phylum level. Specimens were grouped by sponge species and ordered by increasing depth from left to right, Geodia barretti (Gb, 407–1462 m), Stryphnus fortis (Sf, 483–1476 m) and Weberella bursa (Wb, 244–1271 m).

Article Snippet: The barcoded 16S rRNA gene amplicons were sequenced at GATC Biotech AG (Constance, Germany; now part of Eurofins Genomics Germany GmbH) by Illumina Miseq sequencing.

Techniques: