Microbiome sequencing is a method of decoding the genetic material of all the microorganisms in a sample: bacteria, virus, fungi and protists. There are two major methods that are used: whole genome shotgun sequencing (WGS) and amplicon sequencing. WGS is an unbiased method that detects all the genetic material in the sample.
Amplicon sequencing targets a portion of the genetic material that is shared among the microorganisms but contains regions of variability that allow their identification. WGS can also identify functional genes in the sample whereas amplicon sequencing cannot.
Identification of microorganisms in a sample by sequencing allows several advantages over traditional culturing approaches. These include:
CosmosID offers several sequencing solutions, including amplicon sequencing, WGS, isolate sequencing, and metatranscriptomics. The CosmosID team has extensive experience in supporting the design and execution of metagenomics projects for both industry and academia for a wide range of applications, using both human and non-human sample types.
Amplicon sequencing has been an important methodology for microbiologists and microbiome researchers for decades, enabling the generation of whole community analysis of bacteria or fungi in any complex sample.
For bacteria, the 16S rRNA gene is amplified and for fungi the ITS gene is amplified. Both of these genes contain conserved regions which are used to target primers to them to amplify variable regions within that allow taxonomic identification and calculation of relative abundance.
The advantages of an amplicon-based approach are:
Amplicon sequencing also has some drawbacks that should be considered, including:
WGS offers higher taxonomic resolution and accuracy than amplicon sequencing. Here, shotgun sequencing of the entire sample is applied, meaning no amplification step is necessary.
This removes both primer and amplification bias. Further, the entire genome, not only the 16S rRNA gene, can be identified, allowing examination of the functional potential of the microbial community and any virulence, pathogenic, or antimicrobial resistance genes it may contain.
In addition, assembly of the myriad microorganisms can be done to create metagenome-assembled genomes, or MAGS, allowing association of specific genes with the organisms containing them.
This can be done for bacteria, archaea, viruses, protists, and phages within a sample. WGS offers a more comprehensive and accurate overview of the microbiome than amplicon sequencing is capable of, providing more value for the cost.
The general advantages of WGS are:
CosmosID offers two major types of WGS: shallow shotgun and deep sequencing.
Shallow shotgun sequencing can provide all the information above at a lower cost due to sequencing of the samples at a lower depth. Typically, this is 3 – 6 million reads per sample.
This is sufficient coverage to provide a comprehensive overview of the most dominant features of the community. However, if you are interested in low abundance or rare features, you may want to consider deep sequencing.
Advantages of shallow shotgun sequencing include:
Deep shotgun sequencing provides higher read coverage than shallow. This may be anywhere from 10 million reads and higher, depending on the needs of the individual study. Price per sample increases with higher coverage depth.
Some additional advantages are:
Through optimized RNA-based workflows for a range of different sample types, CosmosID offers metatranscriptomic services for both complex samples and microbial isolates for a range of study applications. Here, RNA is extracted from a microbiome sample in order to sequence and characterize the range of genes expressed.
The advantages are:
CosmosID’s team of expert bioinformaticians and software developers have the unique ability to support custom bioinformatics and in silico studies. Example applications include: