Bringing together scale and focus: an introduction to targeted gene expression

Olivia Habern on July 2, 2020

We recently announced the launch of our new Targeted Gene Expression Solution. Find out how targeted gene expression can help you increase the scale of your experiments while decreasing sequencing costs, and explore a use case for single cell CRISPR screens, published by a team of scientists from UCSF.

Capturing a part of the whole: why target enrichment?

The discovery power of single cell and spatial whole transcriptome analysis is well known. Given a complex mixture of cells, or a thinly sliced tissue sample, scientists can profile total mRNA expression from individual or small clusters of cells, deriving meaningful conclusions about cell identities, functional states, and local cellular relationships and interactions, all without the need for pre-selection of known marker genes. The tested value of this analytical method is balanced, however, by the everyday realities of the research process. From the largest institutions and biotech companies to individual laboratories and projects, researchers are trying to push the boundaries of biological discovery and explore their scientific passions while optimizing their experiments with the resources they have.

Imagine you’re a pharmaceutical researcher with several potential drug targets, whether a specific gene or a molecule in a signaling pathway, undergoing ongoing testing. You want to validate your targets quickly and affordably, and don’t necessarily need the comprehensive gene expression data offered by whole transcriptome analysis. Or, imagine you’re looking to profile tissue or blood samples from experimental subjects at multiple time points. Or, perhaps you’re looking to perform single cell sequencing on samples from hundreds of unique subjects for a population-scale study. In both cases, it would be challenging to achieve the desired scale of these studies within a reasonable budget without focusing your sequencing on a selection of gene targets.

This focus and scale is now achievable with Targeted Gene Expression from 10x Genomics. Capturing the best of our single cell and spatial technologies, while making sequencing more accessible and affordable, targeted gene expression allows researchers to toggle between whole transcriptome analysis and target enrichment, leveraging the same final single cell or spatial libraries to amplify a subset of genes of interest. By focusing on a part of the whole, that is, a select number of genes from their samples, they can sequence their genes of interest more deeply, or sequence more samples to scale their studies, while cutting sequencing costs.

But what if you don’t know what genes you want to target? How can you balance the discovery power of transcriptomic analysis with a need for information focused around your specific research questions? To give scientists the flexibility and support their research needs, we’ve developed pre-designed targeted gene expression panels—specifically, Human Pan-Cancer, Human Immunology, Human Gene Signature, and Human Neuroscience—that each incorporate over 1,000 key genes and signaling pathways curated from peer-reviewed publications, databases, and experts across these critical research areas. With these panels, you have access to comprehensive gene sets that can offer rich phenotypic information about your tissue or single cell samples.

Explore these panel options in more detail on our Targeted Gene Expression product page →

An introduction to targeted gene expression technology

So, you think target enrichment could help your research, but how does it work? Explore the technology behind the Targeted Gene Expression Solution from 10x Genomics in this short video:

While there are a number of different methods to achieve targeted gene expression, we chose a hybrid capture approach using biotinylated baits and streptavidin beads to pull them down. Why? In a webinar introducing Targeted Gene Expression, Katie Pfeiffer, Staff Scientist at 10x Genomics, explained the reasoning:

Is the streptavidin method of target capture more specific than PCR methods using primers?

"As we were evaluating different strategies for target enrichment, PCR enrichment was considered. But we found in our proof of concept experiments that the hybrid method of capture was better at recovering the complexity of the initial library. It produced a much more even enrichment of the genes that were present in the parent library. This method is much more amenable to customization as well. When designing multiplex PCRs, if you want to add another 10 or 100 genes, it’s not a trivial exercise to try to design a set of PCRs that will work efficiently together, without creating primer-dimers and other artifacts."

In contrast, a hybridization-based capture approach using gene baits and streptavidin enables effective multiplexing, allowing researchers to sequence thousands of selected transcripts.

Katie also provided insights on the comparative benefits of whole transcriptome analysis and targeted gene expression:

Is targeted gene expression more sensitive than whole transcriptome analysis?

"Sensitivity is a question that is more complicated than it sounds. It’s really about the ability to detect the genes that you’re interested in at the sequencing depths you can achieve. If you can sequence every gene expression library out to its full saturation level, we can’t use targeted gene expression to detect more molecules than whole transcriptome analysis because we are only enriching molecules created during the same whole transcriptome library preparation. In other words, we don’t make any new molecules during targeting, and rightly so.

What targeting allows you to do, however, is access super deep sequencing on only your targets of interest, in a way that is within your budget. For example, we took a glioblastoma sample and sequenced the whole gene expression library out to a significant depth, with 70K reads per cell. But with target enrichment, we were actually able to go much deeper on genes of interest from the sample with a more reasonable sequencing depth of 12K reads per cell. Targeting can enable access to more biological complexity, in that it allows you to sequence deeper and detect more genes and more UMIs from the panel of interest. In that way, it can be more sensitive than the whole transcriptome."

Find answers to more common questions, and explore the Targeted Gene Expression workflow and preliminary datasets in this on-demand webinar. Watch here →

Bringing scale and focus to single cell CRISPR screens

In a study out in Nature Biotechnology, researchers from UCSF demonstrated a technique for single cell CRISPR screens, called direct capture Perturb-seq. This method leverages the Chromium Single Cell Gene Expression Solution with Feature Barcode technology from 10x Genomics to capture multiple distinct sequencing guide RNAs from individual cells, while simultaneously sequencing single cell transcriptomes.

With this approach, the team was able to interrogate the impact of single and double genetic perturbations on the expression patterns of other gene sets—relationships also known as genetic interactions (GIs)—and ultimately on single cell phenotypes. Specifically, they used this approach to study how inhibiting the expression of a set of genes responsible for the biosynthesis of cholesterol impaired the activity of DNA repair genes. This genetic interaction, in turn, led to the accumulation of toxic metabolic intermediates that caused their cell lines to arrest in an unusual replication checkpoint-activated cell cycle. By mapping these complex GIs, they were able to gain a better understanding of which genetic changes and molecules were the true culprits for downstream alterations in the activity of individual cells (1).

While the aim of this study was to optimize single cell CRISPR screens through direct capture of single guide RNAs, the team addressed several additional barriers to optimal CRISPR screens that have relevance to other single cell and spatial sequencing applications. One issue they addressed is scale: as we’ve noted, whole transcriptome single cell and spatial RNA sequencing provides unbiased access to comprehensive cellular and tissue phenotypes, but it also requires significant sequencing resources that may not be realistic for researchers interested in these techniques. Another issue they addressed is gene sensitivity. Gene expression is skewed in a cell, meaning certain genes are more heavily expressed than others. As a result, a small percentage of the total expressed genes could actually take as much as 50% of a researcher's sequencing reads, just because that subset of genes was expressed more than others. In this case, lowly expressed genes that encode important biological molecules, like transcription factors or kinases, could be difficult to measure.

To alleviate these challenges, the research team leveraged target enrichment, an approach that allowed them to sequence only the genes they needed to study or were most interested in exploring further. This was a representative subset of 978 genes that reflected many different transcription states, comparable to the rich information provided by whole transcriptome analysis, and spanned different orders of magnitude of base-level gene expression. Starting with their scRNA-seq library, they enriched the library with biotinylated baits designed to hybridize to all 978 genes, then pulled down those transcripts with magnetic streptavidin beads. By performing post-capture PCR, they were able to amplify these transcripts by over 14-fold, from 6% in an unenriched control to 87% after target enrichment, but at one tenth of the sequencing depth of the original library (1).

Importantly, they were also able to show that hybridization capture on their CRISPR-interference Perturb-seq libraries could reflect the same GIs and relationships to cellular phenotypes revealed by sequencing the entire transcriptome, however at reduced sequencing costs and with the added flexibility to iteratively refine targeted gene subsets in the same experiment from the same library.

You can explore their methods to develop direct capture Perturb-seq in this research snapshot, or in their publication.

Additionally, watch an upcoming webinar hosted by The Scientist with lead author Joseph Replogle to learn more about how Targeted Gene Expression supported their study. Register here →

What will you do with targeted gene expression?

Single cell CRISPR screens provide a powerful use case of targeted gene expression, however there are a number of applications, across research areas, that this technology could support. In fact, this technology could benefit any experimental model that requires scalable sequencing, or deep analysis of specific gene targets.

Whatever application you are interested in, we’re excited to see how targeted gene expression can accelerate your research, remove limitations to the scope and possibilities of your studies, and allow you to gain deeper insights into complex biology than ever before.

Want to get started with the Targeted Gene Expression Solution? Watch this introductory webinar on-demand, and learn more about the technology on our product page →

References:

  1. JM Replogle et al., Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat. Biotechnol. (2020).