Microbiome analysis methods
Several methods are used to analyse microbiota and identify and quantify the members of a microbial community. Analysis can be done at the DNA, RNA, protein and metabolite level.
There are several methods/ techniques for DNA sequencing and RNA sequencing of microbiota and individual microorganisms present in the human microbiome. Next-generation sequencing (NGS) applied to DNA in human feces samples is the predominant method and there are several strategies for NGS of the microbiome.
Metagenomics sequencing using next-generation sequencing (NGS)
Sequencing of microbiomes is sometimes referred to as metagenomics or metagenomic sequencing. The term metagenomics refers to gene-level and genome-level characterization of communities and their members using genomics methods and other “omics” methods that do not require cultivation of organisms.
NGS platforms sequence millions of small DNA fragments in parallel. Bioinformatics uses sequence maps to figure out where the sequenced fragments fit. NGS can sequence entire genomes or target specific regions. Amplicon sequencing targets specific regions by PCR which are then sequenced with NGS.
Ampicon sequencing with NGS
- 16S rRNA. The highly conserved region of the genome that codes for 16S ribosomal RNA in bacteria and archaea is amplified by PCR and sequenced. Specific sequence differences correspond to a taxa at an operational taxonomic unit (OTU). OTUs correspond to organisms at the phyla and genera level and can be less precise at the species level.
- 18S rRNA. Similar to 16S rRNA region used for bacterial and archaea.
- Internal transcribed sequence (ITS). ITS sequences ITS1 and ITS2 are used for amplicon sequencing and identification of fungal DNA.
Whole genome sequencing with NGS
- Whole genome shotgun sequencing (wgs) also called metagenomic shotgun sequencing. All genes in all organisms within the sample are sequenced. Random primers are used to sequence regions of the genome in overlapping reads which are assembled by computers. Using WGS, taxa can be defined at the species level. WGS is more expensive than 16S rRNA amplicon sequencing.
Metagenomics sequencing companies
Metagenomics computational analysis
Metatranscriptomic analysis is concerned with transcribed genes, which provides a snapshot of active genes within a microbial community.
- meta-total RNA sequencing (MeTRS). MeTRS requires a reverse transcription reaction step to convert it into cDNA for sequencing.
- RNA-Seq with NGS. Conversion of mRNA to cDNA is followed by NGS.
- SSU rRNA-based RNA-seq. The target is small subunit rRNA.
- MALDI-TOF mass spectrometry. Matrix assisted laser desorption ionization (MALDI) is a form of mass spectrometry that is used to generate a protein mass fingerprint (PMF) which is compared to a database. Mass spectrometry is a technique that uses ionization to categorize chemical compounds based on their mass to charge ratio. Microbial applications usually use time of flight (TOF) analyzers to measure mass.
- LC‐MS/MS (liquid chromatography combined with mass spectrometry)
- Shotgun metaproteomics
Metaproteomics sofware tools
Metabolomic research is concerned with the metabolic profile including identification and classification of metabolites produced by microbes and deducing the biochemical pathways of those metabolites. The main technologies used in metabolomics are mass spectrometry and nuclear magnetic resonance spectroscopy. Metametabolomics is the analysis of metabolites in a community of microbes.
- GC-MS (Gas Chromatography Mass Spectrometry)
- Tandem Mass Spectral Library (NIST)
- Wiley Registry of Mass Spectral Data
Human gut microbiome components
Bacteria, archaea, yeast, fungi, protists, helminths and viruses are all found in the human gut.
- Many commensal and pathogenic gut bacteria possess invertons which help them colonize and/or contribute to persistance of antibiotic resistance
Gut microbiome catalogs and standards
- MetaHIT (Metagenomics of the Human Intestinal Tract)
- Human Microbiome Project (HMP)
- International Human Microbiome Standards (IHMS) project
- The Microbiome Quality Control project
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- Monsanto Growth Ventures
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