Are you a company, lab or researcher planning a new microbiome study?  If so, you are probably considering whether to conduct 16S rRNA gene sequencing or shotgun metagenomic sequencing. Although 16S rRNA gene sequencing has been more commonly used for microbiome studies to date, shotgun metagenomics is becoming more accessible and popular in microbiome research. However, each method has its pros and cons which should be considered before you decide which sequencing method to use. Here is your one-stop guide to 16S rRNA gene sequencing vs shotgun sequencing to help you generate the best data for your research.

Table of contents

• 16S rRNA sequencing vs shotgun sequencing: the basics
  • 16S rRNA sequencing
  • Shotgun metagenomic sequencing
• 16S rRNA sequencing vs shotgun sequencing: head to head
• Factors to consider
  • Cost
  • Data depth: Genus, species, strain?
  • Bacteria? Fungi? Viruses?
  • Composition vs function
  • Sample type
  • Analysis capabilities
• Some study examples
• Need help?

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Microbiome Sequencing: The Basics

In the year 2000, it cost $100,000,000 to sequence the entire human genome, whilst in 2020 it costs about $1000. These rapid developments in sequencing technologies, which have made sequencing much faster and cheaper, are largely responsible for the fascinating advances in microbiome research in recent times. However, there are now different types of sequencing options available for microbiome studies which are accessible to all researchers, companies and industries interested in microbiome research. So whether it is human, animal or environmental microbiomes that you are interested in, it is important to understand and consider the pros and cons of 16S rRNA vs shotgun metagenomics.

16S RRNA Sequencing

A form of amplicon sequencing, 16S rRNA gene sequencing targets and reads a region of the 16S rRNA gene which is found in all Bacteria and Archaea, meaning this type of sequencing can only identify these types of microorganisms. Other types of amplicon sequencing can identify other microorganisms, such as ITS sequencing for fungi or 18S sequencing for protists. The process of 16S rRNA gene sequencing involves a few simple steps:

1. Extract DNA from your sample
2. Perform PCR on your DNA sample to amplify one or more selected hypervariable regions (V1-V9) of the 16S rRNA gene, as well as adding molecular ‘barcodes’ to each cleaned DNA sample (to multiplex multiple samples)
3. Clean up and size select your amplified DNA to remove impurities
4. Pool samples together in equal proportions
5. Library quantification
6. Sequence pooled samples

The output of 16S rRNA sequencing provides sequencing ‘reads’ (strings of DNA sequence) that can be analysed using a number of basic bioinformatic steps, which when combined together are known as ‘pipelines’. These bioinformatic pipelines remove sequencing errors or dubious reads in order to ‘clean’ the data which can then be aligned to microbial genomic databases in order to accurately identify and profile the bacteria (and archaea) that were present in the samples. A number of 16S sequencing pipelines are available (QIIME, MOTHUR, USEARCH-UPARSE) some of which have extensive tutorials and online user interfaces in order to support researchers without bioinformatics expertise.

Shotgun Metagenomic Sequencing

Shotgun metagenomic sequencing involves randomly breaking (‘fragmenting’) DNA into many small pieces, much like a shotgun would break something up into many pieces. These fragmented pieces of DNA are then sequenced and their DNA sequences are stitched back together using bioinformatics to identify the species and genes present in the sample. Unlike 16S rRNA sequencing, shotgun metagenomic sequencing can read all genomic DNA in a sample, rather than just one specific region of DNA. For microbiome studies, this means that shotgun sequencing can identify and profile bacteria, fungi, viruses and many other types of microorganisms at the same time. As genomes are sequenced, it is also possible to identify and profile microbial genes that are present in the sample (the metagenome), which provide additional information about microbiome functional potential. The process for metagenomic sequencing involves a few extra steps compared to 16S rRNA sequencing (these steps can vary slightly depending on the methods used):

1. Extract DNA from your sample
2. Tagmentation, a process which cleaves and tags(insert) DNA with adapter sequences, priming the fragmented DNA for ligation of molecular ‘barcodes’)
3. Clean up your fragmented DNA sample to remove tagmentation reagent impurities
4. Perform PCR to amplify tagmented DNA samples, as well as adding molecular ‘barcodes’ to each sample.
5. Size selection, and clean up the DNA to remove impurities after the PCR steps
6. Pool samples together in equal proportions
7. Library quantification of the pooled samples
8. Sequence pooled samples

Shotgun sequencing reads require more complex bioinformatics methods in order to analyse results. Shotgun metagenomics bioinformatics pipelines also perform quality filtering steps after which the cleaned sequencing data can either be assembled to create partial or full microbial genomes (using pipelines such as Megahit) or aligned to databases of microbial marker genes (using pipelines such as MetaPhlAn and HUMAnN). Many of these pipelines now also have online tutorials and user interfaces in order to assist those without bioinformatics expertise with analyses. The final results provide details of the relative abundances of bacteria, fungi, viruses and other microbes in the sample as well as the relative abundances of curated lists of microbial genes (e.g. metabolic or antibiotic resistance genes).

16S rRNA Sequencing vs. Shotgun Sequencing: head-to-head

As explained above, shotgun metagenome sequencing provides more information than 16S rRNA sequencing. However, shotgun metagenome sequencing has some limitations. The table below outlines the relative merits of both types of microbiome sequencing:

Factors to consider

Cost

Although shotgun metagenome sequencing provides much more data than 16S rRNA gene sequencing, you will have to pay for that extra data. Shotgun metagenome sequencing is usually at least double to triple the cost of 16S rRNA sequencing, although costs are falling continuously. In order to work around this data vs cost dilemma, some researchers conduct 16S rRNA gene sequencing on all of their samples in addition to shotgun metagenomic sequencing on a small subset of samples in their study. The cost per sample of sequencing will always depend on the depth of sequencing. Deeper sequencing usually means more data and analysis options. In recent years however, a new variation of shotgun metagenome sequencing, termed shallow shotgun sequencing, has managed to bridge the gap between sequencing data and cost. By combining many more samples into a single sequencing run and using a modified protocol that uses less reagents for sequencing library preparation, shallow shotgun sequencing is able to provide >97% of the compositional and functional data obtained using deep shotgun metagenomic sequencing at a cost similar to 16S rRNA gene sequencing. “Shallow” metagenomics is best suited for studies that benefit from the statistical power afforded by a high number of replicates. In its current form, shallow metagenomics is most reliable if it is used for sample types with a high microbial-to-host DNA ratio (e.g. fecal samples).

Taxonomic resolution: Genus, species, strain?

16S rRNA gene sequencing is generally limited to identifying bacteria at the genus level (e.g. Bifidobacteria). Shotgun metagenomic sequencing on the other hand can identify bacteria and other microorganisms at a species (e.g. Bifidobacterium longum) or sometimes even strain level (e.g. Bifidobacterium longum 35624) by profiling single nucleotide variants in metagenomic data. Therefore, for broad profiling of bacterial microbiomes, 16S rRNA sequencing would be sufficient. However, if it is necessary to look a little more deeply at the species and strains within your microbiome of interest, shotgun metagenomic sequencing will be more powerful.

Taxonomic coverage: Bacteria? Fungi? Protists? Viruses?

As discussed above, 16S rRNA sequencing targets a gene that is only present in bacteria and archaea. Although bacteria make up a majority of human microbiomes, there is growing interest in the fungal, viral and eukaryotic proportions of human and other microbiomes. If you are only interested in bacteria, 16S rRNA gene sequencing is sufficient; however, if you are interested in multiple microbial kingdoms, shotgun metagenome sequencing will be more suitable. It is important to consider, however, that the ability to identify bacteria, viruses and eukaryotic microorganisms simultaneously and accurately will strongly depend on the DNA extraction method (e.g. RNA viruses cannot be detected in DNA extract) used on your sample and bioinformatics pipelines used.

Composition vs function

Microbiome research is now moving beyond profiles of microbial taxa in a sample (taxonomic composition). In fact, evidence from large human microbiome studies suggest that functional metagenomic data may provide more power for identifying differences between ‘healthy’ and ‘diseased’ microbiomes. 16S rRNA gene sequencing cannot directly profile microbial genes/functions, however some bioinformatic tools (PICRUSt) are available to predict microbiome function with 16S rRNA gene data. Shotgun metagenome sequencing, on the other hand, can provide comprehensive data on microbial gene content. Therefore, if you are interested in microbiome functional profiles (e.g. antibiotic resistant or carbohydrate degrading genes), shotgun metagenomic sequencing is the more suitable choice. The caveat is that current databases are limited in identifying many functional genes.

Sample type

As 16S rRNA sequencing uses PCR to amplify a specific region of DNA, there is little chance of amplification from the ‘host’ DNA. Shotgun metagenomic sequencing, on the other hand, sequences all the DNA in a sample meaning that non-microbial reads may obscure the microbiome results. This is especially apparent in human microbiome studies using samples that may contain lots of human DNA, such as skin swabs or cheek swabs when looking at the skin and oral microbiomes. For such samples, 16S rRNA gene sequencing may be more suitable.

Analysis capabilities

Although shotgun metagenomic sequencing provides more data than 16S rRNA gene sequencing, the data potential can only be used with the appropriate tools and analysis. Metagenomic sequencing data is complex and therefore it requires more powerful computers, time, and expertise to generate meaningful results from large datasets. 16S rRNA gene sequencing, however, generates simpler data that can be analysed more easily by non-experts. Before deciding on 16S rRNA sequencing vs shotgun metagenomic sequencing, it is important to consider the bioinformatic analysis capabilities available to you. Microbiome Insights have a team of bioinformatic experts that are happy to help with your 16S rRNA or shotgun metagenome sequencing studies.

Databases

As 16S rRNA sequencing has been more commonly used in microbiome studies to date, there are a number of well-curated databases available to identify microorganisms present in your sample. On the other hand, there is currently a lack of full reference genomes of certain microbial species in databases used for shotgun metagenome sequencing. Therefore, in microbiome samples that that have not been previously well characterised (e.g. soil or marine samples), shotgun metagenome sequencing may actually identify fewer taxa than 16S rRNA gene sequencing. Full microbial genomes are continuously being added to these databases however, meaning that shotgun metagenome sequencing will be able to identify these understudied microorganisms as efficiently as 16S rRNA gene sequencing in the future.

Study Examples

Example Study A - Assessment of the bacterial microbiome of Amazonian soil

As discussed above, bacterial 16S rRNA databases may be more suitable for identifying rare or understudied taxa as corresponding full reference genomes may not currently be available for these species. In this example study, 16S rRNA sequencing may provide more taxonomic resolution than metagenomic sequencing.

Example Study B – Changes in microbiome composition and antimicrobial gene carriage following fecal transplant

The primary advantage of metagenomic sequencing is its ability to provide functional microbiome data relating to microbial genes. In this study example, metagenomic sequencing would be the more suitable choice as it would provide simultaneous assessment of the changes in the gut microbiome composition in addition to detailed profiling of antimicrobial resistance genes and their hosts.

Example Study C - Daily fluctuations in gut microbiome following 2 week dietary fiber intervention

Although dietary interventions often change gut microbiome composition, in certain instances changes are only evident at a functional level. In this example study, shallow shotgun sequencing would be a powerful approach to assess both compositional (e.g. species or strain changes) and functional differences in the gut microbiome following a dietary intervention at a cost similar to that of 16S rRNA sequencing. 

Need more advice?

Microbiome Insights provides 16S rRNA sequencing as well as shallow and deep shotgun metagenome sequencing services, plus extensive bioinformatic expertise. See all our microbiome sequencing services here.

If you still have questions about your microbiome study and the type of sequencing to use, the Microbiome Insights team will be happy to help. 

Are you planning a microbiome study? 

We created this guide for anyone interested in studying microbial communities. It covers all the key steps from study design and execution through to analysis and interpretation, including many useful resources and supporting documents.