This repo would serve as beginner's path for metagenomics, while myself a beginner trying to figure out the path and constantly pushing resources and pipline materials. If your'e new to this subject like me start by reading below and move to moving picture followed to other projects.
The contents provided in the repository are not made by generative ai, they are reflection of my understanding towards the subject, down below the content may have been undergone asthetic enhancement using ai without changing any contents provided.
Amplicon sequencing is a method of targeted next generation sequencing (NGS) allowing researchers to analyze genetic variations in specific genomic regions using polymerase chain reaction (PCR) primers designed to amplify a region (regions) of interest in a genome.
The 16S ribosomal rRNA gene in prokaryotes and 18S rRNA gene in eukaryotes is considered as molecular marker for distinguishing.
16S rRNA gene contains 10 highly conserved regions and 9 regions that are variable. In 16S rRNA amplicon sequencing of bacteria, the highly conserved region serves as primer while the variable regions helps in identifying bacterial taxas.
The amplified 16S rRNA gene can then be sequenced on one of numerous platforms including:
- 454-pyrosequencing
- Illumina Hiseq and Miseq
- Ion Torrent
All files generated by QIIME 2 are either .qza or .qzv files, and these are simply zip files that store your data alongside some QIIME 2-specific metadata.
.qzastands for QIIME zipped artifacts, which are intermediary files in an analysis workflow..qzvstands for QIIME zipped visualization, which helps in providing interactive visualizations. These files can be viewed in QIIME2 View
QIIME 2 artifacts are assigned exactly one artifact class, which indicates the semantics of the data (its semantic type) and the file format that is used to store it inside of the .qza file.
QIIME 2 was designed to automatically document analysis workflows for users, ensuring that their bioinformatics work is reproducible.
To achieve this, each QIIME 2 command is recorded when it is run, and that information is stored in all Artifacts and Visualizations that are created.
You can view the data provenance at QIIME2 View by simply uploading the .qza or .qzv file.
Plugins define actions, which are the individual commands that you’ll run in an analysis workflow. Three types of actions can be defined by plugins:
- Methods: take one or more artifact to generate artifacts.
- Visualizers: take artifact(s) to produce
.qzvfiles. - Pipelines: take artifacts and parameters as input, and produce
.qzaand.qzvfiles.
Steps of the workflow:
- Importing the raw sequence — creation of first
.qzafile from FASTQ file - Demultiplexing
- Denoising and Clustering
- Taxonomic Classification
Multiplexing refers to sequencing different samples together, with barcodes attached on one or both ends of each sequence.
Demultiplexing is the process of detecting the barcode sequences and mapping them back to the sample they belong.
These barcode info will be available in the sample metadata file, which can be given as input during demultiplexing.
Denoising removes low-quality reads. Denoising methods available are DADA2 and Deblur, which produce Amplicon Sequence Variants (ASVs).
Clustering helps in removing chimers or similar sequences and replicates them into one representative — known as OTU (Operational Taxonomic Unit) picking.
The outputs are:
- A
FeatureTable[Frequency]artifact - A
FeatureData[Sequence](representative sequences) artifact
- R. Lanza & A. Atala (Eds.), Molecular Biology, Academic Press, 2020. Link
- Paragon Genomics. What is Amplicon Sequencing? Link
- Ellermann, M., Carr, J.S., Fodor, A.A., Arthur, J.C., & Carroll, I.M. (2019). Characterizing and Functionally Defining the Gut Microbiota: Methodology and Implications. In: The Gut-Brain Axis (Chapter 2), Academic Press.
- QIIME2 Docs. Getting Started with QIIME 2 Amplicon Sequencing. Link