One consequence of the rapid evolution of sequencing technologies is that every few years, microbiome researchers need to re-evaluate which genomics technology is the most effective and informative use of their limited budgets. That time has once again arrived, as the highly accurate synthetic long-read sequencing technology developed by Loop Genomics changes the balance of pros and cons for both 16S sequencing (and 18S sequencing for fungi) and shotgun metagenomics approaches, increasing the depth of information researchers can uncover with both methods.
How has Loop changed the sequencing equation?
The big change with Loop technology is that the phrases “long-read” and “highly-accurate” are both true and backed by a great deal of data.
See data and the interactive reports included in Loop Genomics' 16S Long Read Sequencing Service for a run with 8 single-species samples from ATCC and a run on a ZymoBIOMICS Microbial Community Standard (the same community is sequenced as 24 separate samples, demonstrating excellent reproducibility ).
Our technology combines the cost-effectiveness of Illumina short-read sequencing with clever biochemistry and bioinformatics to deliver contigs that are megabases in length and have lower error rates than conventional Illumina sequencing. In addition, our technology introduces no or very low PCR bias (depending on the kit/service) for highly accurate molecular abundance measurements. The implications for metagenomic studies are very exciting, and the bottom line is that you can get high-quality and affordable taxonomic assignment with 16S/18S sequencing and you only need to turn to shotgun metagenomics if you need information on other genes, such as for metabolic profiling. Here's why:
For 16S sequencing, getting accurate long-reads means that instead of choosing a few variable regions to sequence you can get complete sequence for the majority of 16S molecules in your sample (as much as 90% of the molecules).
And, unlike conventional 16S sequencing where you don’t know if the sequence in one variable region came from the same molecule as the sequence in a different variable region, you’ll get the extra taxonomic assignment power of a long contiguous stretch of DNA. The result is assignment of over 99% of full-length molecules at the species or genus level, with zero false-positive assignments.
You’ll also be able to measure relative species abundance so you can understand the diversity of the microbiome under study, and even track population changes over time or in response to different environments.
For 18S sequencing to study fungal communities, the same advantages you get with Loop for 16S sequencing also apply—the 18S-ITS1-ITS2 LoopSeq Kit and Service provide sequence over this entire 2.5 kb region for more detailed taxonomic assignment. You also get accurate abundance measurements and low error rates.
For shotgun metagenomics, getting accurate long-reads means you can obtain more information about novel microorganisms without the need for a reference genome, revealing the sequence sequence of complete genes or even operons. You’ll also get accurate abundance measurements for each contig for a detailed understanding of the metabolic potential of the microbiome under study.
When should I choose 16S LoopSeq/18S-ITS1-ITS2 LoopSeq versus Whole Genome LoopSeq for shotgun metagenomics?
With the advances brought by Loop sequencing technology, choosing between 16S and shotgun metagenomics still depends on your research questions and budget, but the tradeoffs are now lower than with conventional short-read sequencing.
Use 16S sequencing or 18S sequencing when you want to understand the identity and/or relative species abundance of a microbial community and you are not interested in the sequence of other genes. With the accuracy and long read-length of Loop's technology, there's no need to do shotgun metagenomics for high quality taxonomic assignment.
16S/18S sequencing is also a good approach for low-biomass communities, such as from skin, as Loop’s technology is not subject to the same levels of background interference from host DNA due to the way the sequencing library is prepared.
Use Whole Genome LoopSeq for shotgun metagenomics when you want to learn more about the genomes of the organisms in your sample such as for functional profiling. Shotgun metagenomics is also a good choice if you’re interested in learning more about novel organisms that might not be in a reference database. Depending on the goals of your study, you can maximize budget by choosing to focus on counting the widest number of molecules (sequence many barcodes at a lower read-depth) or species discovery (sequence fewer barcodes at a deeper read-depth) or both.
Summing up—with Loop’s sequencing technology, your choices are all good
The most exciting part about revisiting the 16S/18S vs shotgun metagenomics question right now is that with Loop’s technology both choices deliver enhanced information compared to conventional short-read sequencing. Learn more about our kits and services and advance your insights into microbial communities.