Journal: bioRxiv

Article Title: In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota

doi: 10.1101/073965

Figure Lengend Snippet: In-field 16S rRNA gene amplicon sequencing of cryoconite bacterial communities showing (A) phylum and family level taxonomic distribution of (B) open and closed cryoconite holes sampled on Vestre Brøggerbreen. The top image shows a “closed” cryoconite hole where the snow and superimposed ice cover has been displaced to reveal the hole, while the middle image shows a seasonally-open cryoconite hole. The bottom image shows the arrangement of equipment for DNA extraction, 16S rRNA gene PCR and nanopore sequencing in a field lab.

Article Snippet: Samples were transferred to the NERC Arctic Research Station Ny Ålesund within three hours and DNA extracted and quantified as described above within the field lab. Bacterial 16S rRNA genes were amplified from 50 ng DNA per sample diluted to 10 µL nuclease free water in 50 µL with 1 × LongAmp Taq master mix (New England Biolabs, Inc), 1 µL 16S rRNA gene barcoded primer (ONT-SQK-RAB-201, Oxford Nanopore Technologies, Ltd) and 14 µL nuclease free water.

Techniques: Amplification, Sequencing, DNA Extraction, Nanopore Sequencing

Journal: bioRxiv

Article Title: In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota

doi: 10.1101/073965

Figure Lengend Snippet: multivariate discrimination of cryoconite bacterial communities revealed by in-field 16S rRNA gene amplicon sequencing. Analyses are performed with data aggregated to phylum/proteobacterial class (A-B) or family-level taxa (C-D) with Hierarchical Cluster Analysis (HCA, subpanels A, C,) or Principal Cooordinates Analysis (PCoA, B,D,). Closed holes (VB1-3) and open holes (VB5-6) are ordinated by multivariate analysis of of fourth-root transformed Bray-Curtis distances of phylotype relative abundances.

Article Snippet: Samples were transferred to the NERC Arctic Research Station Ny Ålesund within three hours and DNA extracted and quantified as described above within the field lab. Bacterial 16S rRNA genes were amplified from 50 ng DNA per sample diluted to 10 µL nuclease free water in 50 µL with 1 × LongAmp Taq master mix (New England Biolabs, Inc), 1 µL 16S rRNA gene barcoded primer (ONT-SQK-RAB-201, Oxford Nanopore Technologies, Ltd) and 14 µL nuclease free water.

Techniques: Amplification, Sequencing, Transformation Assay

Journal: bioRxiv

Article Title: In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota

doi: 10.1101/073965

Figure Lengend Snippet: Benchmarking nanopore 16S rRNA gene amplicon sequencing of cryoconite bacterial communities by comparison with laboratory-generated data. (A) Phylum and family level taxonomic distribution of cryoconite bacterial communities and (B) phylum and (C) family level taxon distributions used for principal coordinates analysis of fourth-root transformed Bray-Curtis distances of phylotype relative abundances discriminate between Arctic and alpine cryoconite communities. Arctic glaciers (blue, AB, ML,VB) and Alpine glaciers (red, GF, RM) are clearly ordinated.

Article Snippet: Samples were transferred to the NERC Arctic Research Station Ny Ålesund within three hours and DNA extracted and quantified as described above within the field lab. Bacterial 16S rRNA genes were amplified from 50 ng DNA per sample diluted to 10 µL nuclease free water in 50 µL with 1 × LongAmp Taq master mix (New England Biolabs, Inc), 1 µL 16S rRNA gene barcoded primer (ONT-SQK-RAB-201, Oxford Nanopore Technologies, Ltd) and 14 µL nuclease free water.

Techniques: Amplification, Sequencing, Comparison, Generated, Transformation Assay

Journal: bioRxiv

Article Title: In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota

doi: 10.1101/073965

Figure Lengend Snippet: Benchmarking nanopore 16S rRNA gene amplicon sequencing of cryoconite bacterial communities by comparison with laboratory-generated data. Correlation of log relative abundances between (A) the ratio of Alphaproteobacteria and Betaproteobacteria (B) Cyanobacteria to Alphaproteobacteria:Betaproteobacteria and (C) key taxonomic groups revealed using nanopore and pyro-sequencing of 16S rRNA genes. Positive, significant or highly significant Pearson r correlations are observed for each taxon.

Article Snippet: Samples were transferred to the NERC Arctic Research Station Ny Ålesund within three hours and DNA extracted and quantified as described above within the field lab. Bacterial 16S rRNA genes were amplified from 50 ng DNA per sample diluted to 10 µL nuclease free water in 50 µL with 1 × LongAmp Taq master mix (New England Biolabs, Inc), 1 µL 16S rRNA gene barcoded primer (ONT-SQK-RAB-201, Oxford Nanopore Technologies, Ltd) and 14 µL nuclease free water.

Techniques: Amplification, Sequencing, Comparison, Generated

Journal: bioRxiv

Article Title: In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota

doi: 10.1101/073965

Figure Lengend Snippet: Benchmarking nanopore16S rRNA gene amplicon sequencing by sequencing mock communities using in-field protocols. Panel (A) shows the expected taxonomic distribution of The ZymoBIOMICS Microbial Community Standard for each sample, spiked in triplicate with Micrococcus luteus NCTC2665 genomic DNA to afford theoretical relative abundances of 16S rRNA genes in the range of 0-1.2% of the total bacterial community. Panel (B) shows shows the observed data where all reads assigned to genus level (excepting the poorly resolvable Enterobacteriaceae while Panel (C) shows all reads at the family level. The expected and observed Micrococcus luteus spike is highlighted in yellow.

Article Snippet: Samples were transferred to the NERC Arctic Research Station Ny Ålesund within three hours and DNA extracted and quantified as described above within the field lab. Bacterial 16S rRNA genes were amplified from 50 ng DNA per sample diluted to 10 µL nuclease free water in 50 µL with 1 × LongAmp Taq master mix (New England Biolabs, Inc), 1 µL 16S rRNA gene barcoded primer (ONT-SQK-RAB-201, Oxford Nanopore Technologies, Ltd) and 14 µL nuclease free water.

Techniques: Amplification, Sequencing

Journal: Current Biology

Article Title: Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes

doi: 10.1016/j.cub.2021.03.056

Figure Lengend Snippet: FISH Wolbachia visualization in the ovaries Wolbachia was primarily located in the ovarian follicles (A–H). Colored boxes indicate area of magnification for subsequent images. Within the same ovary, some ovarian follicles are sparsely infected with Wolbachia (E and magnification in G), while others have a heavy infection (C, D, and H; E and F). Asterisks indicate infection in the secondary follicles. Wolbachia was imaged with an Alexa 590-labeled probe targeting the Wolbachia 16S rRNA gene (red), and DNA was stained with DAPI (blue). No probe control images (I–L) show no fluorescent signal (images in I and J and in K and L are for two separate individuals). FISH analysis revealed that 9/16 individuals were Wolbachia infected. See also Figure S2 .

Article Snippet: The microbiomes of selected individual mosquitoes were analyzed using barcoded high-throughput amplicon sequencing of the bacterial 16S rRNA gene (with library preparation and Illumina sequencing carried out commercially by Source Bioscience, Cambridge, UK).

Techniques: Infection, Labeling, Staining, Control

Journal: Current Biology

Article Title: Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes

doi: 10.1016/j.cub.2021.03.056

Figure Lengend Snippet: Wolbachia strain densities and relative abundance in the mosquito microbiome (A) Normalized Wolbachia strain densities measured using qPCR of the conserved Wolbachia 16S rRNA gene. A synthetic oligonucleotide standard was used to calculate Wolbachia 16S rRNA gene copies per nanogram of total DNA using a 10-fold serial dilution standard curve. p values from t tests are shown to indicate significant differences. (B) Relative Wolbachia abundance in the mosquito microbiome. Taxonomic abundance of bacterial ASVs within the 16S rRNA microbiomes of An. demeilloni and An. moucheti using QIIME 2 was used to determine Wolbachia percent abundance of total 16S rRNA bacterial load, indicated through box-and-whisker plots (GraphPad Prism 9). See also Figure S3 .

Article Snippet: The microbiomes of selected individual mosquitoes were analyzed using barcoded high-throughput amplicon sequencing of the bacterial 16S rRNA gene (with library preparation and Illumina sequencing carried out commercially by Source Bioscience, Cambridge, UK).

Techniques: Serial Dilution, Whisker Assay

Journal: Current Biology

Article Title: Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes

doi: 10.1016/j.cub.2021.03.056

Figure Lengend Snippet:

Article Snippet: The microbiomes of selected individual mosquitoes were analyzed using barcoded high-throughput amplicon sequencing of the bacterial 16S rRNA gene (with library preparation and Illumina sequencing carried out commercially by Source Bioscience, Cambridge, UK).

Techniques: SYBR Green Assay, Sequencing, Software