NGS-809-16S

Overview

1. Introduction

Assessment of bacterial diversity through sequencing of 16S ribosomal RNA (16S rRNA) genes has been an approach widely used in environmental microbiology, particularly since the advent of high-throughput sequencing technology. Oxford Nanopore Technology (ONT) provides the long reads sequencing that cover the full-length sequence of 16S rRNA gene (V1-V9 regions) through a fast, cheap, and high throughput process. Since all the informative sites of 16S rRNA genes are considered, the full-length 16S rRNA sequences offer a higher level of taxonomic and phylogenetic resolution for bacterial identification [1].

2. Methods

DNA concentration was determined using both NanoDrop spectrophotometers and Qubit fluorometer. Library preparations were conducted using Kits from Oxford Nanopore Technology.

Nanopore sequencing was operated by MinKNOW software version 24.02.16. Basecalling was performed using Dorado version 7.3.11 with high-accuracy model [2]. The quality of FASTQ files were visualized using NanoPlot, and quality filtering was performed using NanoFilt [3][4].

Filtered reads were classified using Centrifuge classifier [5]. Bacteria and Archaea index was built using NCBI 16S RefSeq database (https://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/). Downstream analysis and visualizations were performed using Pavian (https://github.com/fbreitwieser/pavian), Krona Tools (https://github.com/marbl/Krona), and RStudio using R version 4.3.3 (https://www.R-project.org/).

3. Workflow

3.1. Experimental Workflow

3.2. Bioinformatics Workflow

4. References

Bahram M, Anslan S, Hildebrand F, Bork P, Tedersoo L. 2018. Newly designed 16S rRNA metabarcoding primers amplify divers and novel arhaeal taxa from the environment. Environ. Microbiol. Rep. 11(4): 487-494. https://doi.org/10.1111/1758-2229.12684.
Wick RR, Judd LM, Holt KE. 2019. Performance of neural network basecalling tools for Oxford Nanopore sequencing. Genome Biol. 20:129. https://doi.org/10.1186/s13059-019-1727.
de Coster W, D'Hert S, Schultz DT, Cruts M, van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34(15):2666-2669. https://doi.org/10.1093/bioinformatics/bty149.
Nygaard, A.B., Tunsjø, H.S., Meisal, R. and Charnock, C., 2020. A preliminary study on the potential of Nanopore MinION and Illumina MiSeq 16S rRNA gene sequencing to characterize building-dust microbiomes Scientific Reports 10.1 (2020): 1-10. https://doi.org/10.1038/s41598-020-59771-0.
Kim D, Song L, Breitwieser FP, Salzberg SL. 2016. Centrifuge: Rapid and sensitive classification of metagenomic sequences.Genome Res.https://doi.org/10.1101/gr.210641.116.

Sample Information and QC

1. Sample Information Table

Sample QC NGS-809-16S.

2. Agarose Gel Electrophoresis Photo of PCR Product (Amplification of gDNA with Primer 16S 27F – 1492R)

Sequencing QC

The quality of FASTQ files were assessed by NanoPlot.

Sample ID	Raw		Filtered
Sample ID	QC Stats	Full Report	QC Stats	Full Report
G_Gk1	view	download	view	download
G_Gk2	view	download	view	download
G_Bt1	view	download	view	download
G_Bt2	view	download	view	download
G_KP1	view	download	view	download
G_KP2	view	download	view	download

Results

1. Reads Classification

Reads classification table contains the output of reads classification by Centrifuge software. Click "CSV" button to download the raw data and open it in another viewer, such as Microsoft Excel.

Please select sample:

2. Top 10 Barplot

Top 10 of relative abundance from each taxonomic level (Phylum to Species).

3. Abundance

Total count & relative abundance of each taxonomic level are presented in the CSV table. Download each file and open with Excel application.

Rank	Count	Relative
Phylum	view	view
Class	view	view
Order	view	view
Family	view	view
Genus	view	view
Species	view	view

4. Krona Visualization

Krona visualization allows us to intuitively explore the relative abundances within the complex hierarchies of metagenomic classifications. The count in Krona was based on the numReads from Centrifuge Report. Click "snapshot" to convert the chart into a static image.

5. Sankey Diagram

Sankey diagrams are used to visualize microbial species of samples. In this diagram, the arrow width is proportional to the quantity to depict changes over time hierarchy between nodes.

Please select sample:

If the image does not load correctly, press right click -> reload frame.

6. Alpha Diversity

Alpha diversity describes the mean species diversity in sites or habitats at local scale.

richness — Boxplot of Alpha Diversity. Download: full size image - raw data

7. Heatmap

Heatmap depicts taxon relative abundance of each sample using shades of colour to easily identify patterns among samples. Red indicates a taxon with high distribution while black shows low relative abundance. Top 50 most abundant OTUs were shown on the graph.