When new sequencing instruments are released, great leaps in genomics capabilities follow. But did you know that for some applications like 16S microbiome research, gene annotation, or cancer transcriptomics, “new-instrument-level” performance can be achieved with something as simple as an off-the-shelf kit?
That is exactly what the new Kinnex kits for full-length 16S, bulk RNA, and single-cell RNA sequencing deliver.
In this piece, we explore a series of talks given by PacBio experts and third-party scientists as they dive into what Kinnex kits are, how they work, and how they can enable you to access a whole new tier of discovery potential with insights from high-throughput full-length RNA and 16S sequencing.
Even if you don’t own or have immediate access to a PacBio long-read sequencing system, you’ll want to tune in, because sequencing service providers like Zymo Research and others are incorporating Kinnex kits into their workflows –making Kinnex full-length RNA and 16S sequencing data more accessible than you’d think.
For each talk, we’ve got a quick summary, the must-know highlights, and if we’ve piqued your curiosity – links to watch the recordings:
Jump to topic:
What is Kinnex? Kinnex for gene annotation Kinnex 16S services from Zymo Full-length 16S in human gut microbiome research How to multiplex with Kinnex
What Kinnex is –and why it’s a game-changer
Jacob Brandenburg, Plant and Animal Genomics Lead at PacBio, walks viewers through what Kinnex kits are, how they work, and how they change what’s possible for researchers who rely on RNA and amplicon sequencing to advance their science. The discussion spans applications in diverse fields, with a particular emphasis on the utility of full-length 16S reads in microbial ecology and microbiome studies.
Key takeaways:
- The highly accurate long reads provided by PacBio HiFi sequencing are ideal for cutting edge plant and animal genomics and microbiome research.
- Full-length 16S sequencing stands out as the definitive method for more comprehensively resolving clades found in the human gut. This approach is unbiased and boasts a high rate of reads that can be classified down to the species and even strain level with precision.
- The Kinnex full-length RNA and 16S rRNA kits provide researchers with access to exacting information by taking multiple amplicons or cDNA as an input and concatenating them into a single molecule for sequencing. The result is a massive throughput increase of 8 to 12-fold for full-length RNA or 16S rRNA, respectively.
- Full-length 16S rRNA sequencing with the Kinnex kit yields species and strain-level insights that would be difficult to achieve even with shotgun approaches using conventional short-read technologies. With Kinnex, full-length 16S HiFi sequencing is now attainable at a price-point that is highly competitive with inferior partial 16S alternatives.
How Kinnex is setting a new standard in genome annotation of plants and animals at HudsonAlpha
Jeremy Schmutz, the esteemed Co-Director of the HudsonAlpha Genome Sequencing Center and a globally recognized authority in genome sequencing and tool development, takes viewers on a journey through the transformative impact of Kinnex kits. He illustrates how these kits have expanded the horizon for achieving highly accurate gene annotation across a broad spectrum of organisms.
Key takeaways:
- “Kinnex full-length RNA is useful because now we can get 10x-20x the number of full-length cDNAs at a similar cost, move annotation to full-length RNA instead of short-read RNA-Seq, resolve annotations in complex polyploids (even haplotype-specific annotations), and obtain new discoveries in expression space with alternative transcripts for larger and complex genes.”
- Kinnex full-length RNA –run on the Revio system– enabled improvements in annotation completeness compared to the previous version of some plant genome annotations. 5’ ends are more complete, overlapping 3’ ends were corrected, it helped in fixing a broken gene, and it enabled the identification of genes that were missed before.
- Conclusion about Kinnex: “It’s a real excellent advance in the number of full-length cDNAs captured –with 10 to 20x over the Sequel II Iso-Seq with lower cost, so it opens up a lot of opportunities I think to use this data. The overall quality of the cDNA is higher with Kinnex on Revio, we validated it as a drop-in replacement for annotation right now versus the older Iso-Seq versions.”
- “With our improved annotation algorithms, we should be able to provide more accurate structural annotations and I think with great benefits for our tetraploids or complex genomes that we work with on the plant side. I’m super excited to see what we can learn in polyploids or gene families about variation expression and the biology that really drives it in these organisms by being able to sample and identify the full copies of these genes with the Revio and Kinnex that we could never do before with RNA-seq.”
Zymo Research now offers full-length 16S sequencing services powered by Kinnex
Ethan Thai, the Microbiome Operations and Services Manager at Zymo Research, guides viewers through the company’s thorough testing and validation process for the Kinnex 16S kit. He also explains why Zymo Research has decided to include it in their microbiome sequencing services. This presentation is especially relevant for microbiologists seeking advanced 16S data who may not have a PacBio long-read sequencer at their disposal.
Key takeaways:
- Compared to short-read 16S, Zymo’s full-length 16S rRNA services enable you to get a more complete picture of microbiomes with incredible species resolution by capturing the entire 1.6 kb of the microbial 16S gene. Compared to shotgun metagenomic sequencing, PacBio full-length 16S studies are more affordable, achieve comparable taxonomic resolution, have a faster turnaround time, giving researchers the ability to look at a wide array of sample types.
- Based on preliminary data generated using the Zymo fecal reference, outputs showed a dramatic increase from 2.7 million full-length 16S HiFi reads with the monomer approach to a whopping 30 million full-length 16S HiFi reads with Kinnex. “At Zymo we are very excited to see this improvement.”
- Zymo Research scientists set out to test how the Kinnex 16S kit stacks up against traditional monomer 16S in terms of data output. Their findings were impressive: HiFi reads and average reads per sample saw a more than tenfold increase with the Kinnex 16S kit. This robustness test wasn’t just a controlled lab experiment; it was crafted to reflect the diverse array of sample conditions that Zymo Research’s service team deals with regularly. The test included a variety of DNA concentrations and accounted for different types of host DNA, such as human, animal, and plant. It also spanned a broad spectrum of microbial diversity, ranging from low to high biomass samples and included varying degrees of PCR inhibitors. To top it off, microbiome standards were thrown into the mix as a benchmark for quality control.
- Thai concluded that “Now you can enjoy all the perks of this optimized, state of the art workflow from collection to conclusion with PacBio full-length 16S at the same price as 16S with short reads.”
Full-length 16S leads to long sought after insights into gut microbiome development in pre-term infants
Chinnu Jerard of Telethon Kids Institute at Perth Children’s hospital in Australia presents findings from some of recent work on the dynamics of microbiome development in pre-term infants. Jerard’s work, previously constrained by the limited taxonomic resolution of conventional short-read sequencing, saw significant improvement after incorporating full-length 16S sequencing.
Key takeaways:
- The negative impact of pre-term birth on the composition of the infant gut microbiome has been well documented. Current research literature supports supplementation with probiotics to accelerate gut microbiome maturation and reduce intestinal inflammation in extremely pre-term infants.
- Studies that form the basis of this foundational literature are based on conventional short-read 16S sequencing which provides only genus-level taxonomic resolution.
- Meaningful insight into the functional capabilities of these gut microbial consortia requires species and even strain-level resolution.
- PacBio full-length 16S sequencing enabled Jerard to finally examine infant gut microbiomes with critically needed species-level diversity information.
- She found that, “the species-level taxonomy results showed that the probiotic supplementation in pre-term infants aligns their gut microbiome closer to that of full-term infants.”
- “Probiotic use narrows the alpha-diversity gap between preterm and full-term infants over a year …and as a result pre-term babies given probiotics show a microbial composition more closely resembling that of full-term babies.”
How to make Kinnex work for you: a guide to multiplexing and primary analysis
More full-length reads mean more possibilities. In this talk, Jocelyne Bruand, PhD, Senior Staff Scientist of Bioinformatics at PacBio speaks in detail about how Kinnex kits enable more full-length RNA and 16S reads and sample batching/multiplexing options on PacBio long-read sequencing systems.
Key takeaways:
- A major advantage of the Kinnex series of kits is their ability to increase throughput. This boost in read count allows for increased multiplexing capacity and flexibility on each SMRT Cell.
- Using a Kinnex kit, how many samples can be put on a SMRT Cell? For Kinnex full-length RNA on Revio you can do 4 samples with >10M reads each, or 8 samples with > 5M reads each. For Kinnex full-length 16S, you can do 384 samples on a Sequel II/IIe with > ~40k reads each or 1536 samples on Revio with ~30k reads each, or on a Sequel II/IIe with ~10k reads each, respectively.
- Kinnex analysis has been greatly simplified with the release of SMRT Link v13.0, the software used to setup runs on PacBio long-read systems.
- Kinnex full-length RNA sequencing shines by identifying a significantly larger number of known isoforms compared to traditional methods. What’s more, there’s a strong correlation in isoform counts between Kinnex and the standard Iso-Seq approach. Plus, the distribution of transcript lengths is remarkably consistent between Kinnex and regular Iso-Seq, highlighting the reliability and precision of Kinnex for in-depth RNA analysis.
- With Kinnex and traditional Iso-Seq, the way reads are classified stays pretty consistent across various levels of read coverage. Interestingly, as coverage goes up, we see an increase in the proportion of novel isoforms identified. This suggests that the more data we gather, the better Kinnex gets at uncovering new isoforms, maintaining its consistency even as the depth of sequencing increases.
- SMRT Link data streamlines the process by demultiplexing 16S amplicon reads, which can then be seamlessly integrated into existing 16S analysis pipelines, like the HiFi-16S-workflow.
- Analyzing data from the Zymo fecal reference sample (D6323) revealed a strong correlation in genus and species representation between traditional full-length 16S and Kinnex full-length 16S sequencing. The significant increase in reads with Kinnex 16S enhances the detection of lower abundance species. Additionally, when examining the ATCC MSA-1003 sample, a higher correlation with expected species representation was found using Kinnex full-length 16S versus traditional full-length 16S, underscoring Kinnex’s improved sensitivity.
- The increased number of reads provided by Kinnex 16S more effectively captures the diversity of complex samples.
Ready to try Kinnex?
Inspired by what you’ve seen? Are you ready to see what high-throughput, full-length RNA and 16S sequencing can do for your research? We want to hear from you!
Speak with a PacBio scientist