Microbiome research relies on accurate identification and classification of thousands of microbial species in a single biological sample. To date, this has largely been conducted using amplicon sequencing, such as 16S rRNA gene sequencing for the identification of bacteria and archaea. However, shotgun metagenomic sequencing is a powerful tool that has distinct advantages, such as the identification of fungi, viruses, and other microorganisms in addition to elucidation of microbial gene functions. For someone who has never conducted shotgun metagenomic sequencing, it may seem difficult to know where to start.
This guide will explore the principles and applications of shotgun metagenomic sequencing, including sample preparation and data analysis. We will also discuss the limitations and potential pitfalls of shotgun sequencing and provide tips for optimizing your experiment.
Shotgun metagenomic sequencing is a powerful form of DNA sequencing that can identify multiple types of microbes and microbe functions within a biological sample. Unlike amplicon sequencing, which sequences individually selected gene regions such as the 16S rRNA gene found in bacteria and archaea, shotgun metagenomic sequencing involves sequencing all regions of genomic DNA from microorganisms in a sample.
The term “shotgun” sequencing is derived from the process used, whereby the DNA within the sample is fragmented into many small pieces, much like a shotgun would break something into pieces. The small DNA pieces are then sequenced, and their gene sequences are stitched back together using bioinformatics. This allows for the analysis of a wide range of genes and provides comprehensive insights into the microorganisms in the sample and their genetic potential.
Shotgun metagenomic sequencing is used to identify the wide range of microbes and their genetic functions within a particular sample. It therefore has a number of applications in industry, medicine, public health, and more:
Environmental microbiology: Shotgun metagenomic sequencing can be used to identify and classify microorganisms present in soil, water, and air samples, and to gain insights into their functional capabilities and the roles they play in the environment. For example, it has been used to study microbes in soil samples and permafrost, which may give insights into the effects of climate change on microbial life.
Medical microbiology: Shotgun metagenomic sequencing can be used to identify and classify bacteria and archaea present in clinical samples, such as those collected from the human microbiome or from infected tissue. This can help diagnose and treat infections, provide insights into the role of the microbiome in health and disease, or help detect and prevent hospital infection outbreaks.
Food microbiology: Shotgun metagenomic sequencing can be used to identify and classify microorganisms present in food products, such as fermented foods and beverages, or to ensure food safety and quality. It can also help to identify and track food-borne disease outbreaks.
Industrial microbiology: Shotgun metagenomic sequencing can be used to identify and classify microorganisms present in industrial processes, such as those involved in the production of biotechnology products or the treatment of wastewater.
As with all microbiome studies, the collection and storage of your sample is critical to obtaining accurate, reliable, and reproducible gene sequencing results. Samples can be collected in a number of ways, depending on whether they are human fecal samples, soil samples, water samples, swabs or any other type of samples. In general, the three most important factors to consider are:
Once the sample has been collected and prepared, the next step is to extract the DNA from the microorganisms in the sample. This is typically done using a DNA extraction kit, which uses a combination of chemical and physical methods to separate the DNA from other cellular components. There are different DNA extraction kits available, and the choice of kit will depend on the type of sample being analyzed and the specific goals of the experiment. The selection of the DNA extraction kit has a significant impact on the picture of the microbial community that will be observed, and influences the ability to compare studies. In general, all DNA extraction kits include the following steps:
In a few cases, additional pre- or post-treatment steps are needed to break hard-to-lyse structures such as spores, separate specific components of the microbiome such as viruses, or get rid of DNA contaminants such as soil humic acids. This may involve adding enzymes to break down cell walls, using heat to denature proteins, or using physical methods to separate cells from debris. Read our DNA extraction blog for more details about how to optimize DNA extraction for microbiome studies.
Library preparation refers to the series of steps taken to prepare the DNA for sequencing. For shotgun metagenomic sequencing, these steps include the following:
Once the library has been prepared, the next step is to sequence the DNA fragments using a high-throughput sequencing platform. During sequencing, the DNA fragments are randomly amplified and sequenced using a combination of chemistry and optics. The resulting data generates short DNA sequences that can be aligned to databases to identify which microbial species that they belong to and the genes that they encode. A number of different sequencing platforms are available for shotgun sequencing, with sequence length and throughput being the most important factors in the selection of the platform.
Once the sequencing is complete, the next step is to analyze the data to identify and classify the microorganisms present in the sample. This is conducted using bioinformatics pipelines that help to clean up the sequencing data by removing human reads and sequencing errors, and align the cleaned reads to public databases such as the National Center for Biotechnology Information (NCBI) database. There are two primary approaches to data analysis of shotgun metagenomic sequencing, depending on the specific goals of the experiment:
While shotgun metagenomic sequencing is a powerful tool for identifying and characterizing microorganisms, it is important to be aware of its limitations and potential pitfalls. The main advantage of shotgun metagenomics is higher resolution in the taxonomic profiles. In contrast with amplicon sequencing where groups are classified to the genus or species level, shotgun metagenomics allows for species to strain levels. In addition, shotgun metagenomics accesses the genetic potential of the community. On the technical side, since there is no PCR step, no primer bias, copy-number-bias, or PCR artifacts are present. Neither do chimeras occur with shotgun metagenomics.
Cost: Shotgun metagenomic sequencing is more expensive than amplicon sequencing methods such as 16S rRNA gene sequencing. However, depending on how much detail needed on microbial genomes and the sample type, it is possible to perform shallow shotgun sequencing at costs similar to 16S amplicon sequencing.
Bioinformatics expertise: Shotgun metagenomic sequencing requires more computational power as well as expertise in bioinformatics to process and analyse the resulting data. However, publicly available tutorials and pipelines are available for non-experts.
Databases: Depending on the analysis approach, shotgun metagenomic sequencing often relies on comparison to particular databases, and the accuracy of the taxonomic and functional assignments depends on the quality and coverage of the databases. In addition, since amplicon sequencing has a longer history of use, there are more environments for which relevant information may be available if you want to compare your results to those from other studies.
“Contaminating” reads: As shotgun sequencing looks at all genomic DNA within a sample, there is a higher risk of sequencing DNA from unwanted, non-microbial sources. For example, when analysing the skin microbiome from a skin swab, a huge proportion of sequencing reads will come from human DNA and only a small proportion from microbial DNA.
To optimize your shotgun metagenomic sequencing experiments and obtain accurate and reliable results, there are a few key things to consider:
Think about sample selection: Microbiomes can exhibit large temporal and spatial variation over different sampling regions and times. It is therefore essential to select a consistent sample set that is representative of your population or environment.
Create rigorous sample collection protocols: Microbiome samples are at high risk of contamination from other microbes. It is therefore essential to collect, transport, and store your samples as safely as possible to ensure your results are truly representative of the microbiome that you are studying.
Optimize the sequencing coverage: The sequencing coverage, or the number and length of the reads obtained, can significantly impact the accuracy and resolution of the results. It is important to optimize the sequencing coverage to ensure that enough data is obtained to accurately classify and annotate the microorganisms present in the sample. This is particularly important for the identification of different microbial strains or single nucleotide variants. Sequencing depth also controls whether assembly, and all the analyses that depend on it, are possible.
Use negative/positive controls: Use appropriate controls, such as negative controls and DNA standards, to minimize the risk of errors and ensure the accuracy of the results. This will help to confirm if any contamination occurred in your protocol and can identify whether your results are capturing all types of microbes.
Use appropriate data analysis methods: Shotgun metagenomic sequencing analysis relies on a number of different assembly or alignment tools. The choice of these methods will depend on your study and should be considered carefully beforehand.
The team at Microbiome Insights are experts in shotgun metagenome sequencing and provide a full set of services from sample preparation to bioinformatics analysis. If you still have questions about using shotgun metagenome sequencing for your microbiome study, reach out and the Microbiome Insights team will be happy to help.