Enrich for rare targets from complex RNA-Seq libraries—while preserving native transcript abundance signals—with myBaits® Custom RNA-Seq Target Capture kits.
Overcoming Challenges of Total RNA Sequencing
Sequencing of total RNA in a sample (RNA-Seq) is a powerful NGS technique for directly assessing signals of gene expression in a sample. However even with widely-used methods of removing undesired or non-informative transcripts from a sample prior to total RNA-Seq (e.g. rRNA removal), total RNA-Seq samples are still highly complex, and typically require deep sequencing to fully resolve the signal of relatively rare transcripts that are often of major interest to the experiment.
With myBaits Custom RNA-Seq hybridization capture kits, it is easy to design a customized enrichment panel for just your genes of interest, which can then be used to “enrich” those targets from a complex NGS library prior to RNA-Seq. Your genes of interest can themselves be highly complex targets, as it is possible to target anywhere from dozens up to hundreds of thousands of different loci in a single myBaits Custom kit. For example, enriching for all members of a certain gene family of interest, or enriching all genes from a given pathogen genome out of a complex host+pathogen RNA sample. Target capture allows you to reduce per-sample sequencing costs by orders of magnitude while still preserving the essential gene expression signals necessary for downstream analyses.
Benefits of Hybridization Capture with RNA-Seq:
- Enrich for rare transcripts in complex cDNA libraries (e.g. pathogen vs. host)
- Preserve essential transcript abundance / gene expression signals
- Reduce per-sample NGS costs by orders of magnitude
Application: Discover & Quantify NLR Genes with cDNA RenSeq
The Resistance Gene Enrichment Sequencing (RenSeq) workflow allows for the comprehensive study of highly complex disease resistance gene (R gene) families within crop plant genomes. Using comprehensive, customized hybridization capture panels (such as myBaits Custom target capture kits), researchers are able to enrich for diverse “NB-LRR” or “NLR” structural class gene families that confer agriculturally-relevant disease resistance from a variety of pathogens. RenSeq enrichment baits can be used on both DNA-Seq and RNA-Seq libraries in order to dramatically reduce per-sample NGS sequencing costs while providing comprehensive profiling of known and novel NLR regions genome-wide. When RenSeq is performed on a cDNA library prior to RNA-Seq, the enrichment preserves the native transcript abundance signal, and provides a highly cost-effective solution for detecting even low expression of specific NLR genes of interest.
High Accuracy and Reproducibility of myBaits cDNA RenSeq Enrichment. (A) A scatter plot of log2 read counts of NLR genes shows a high correlation between cDNA RenSeq and deep RNA-Seq libraries. (B) Hierarchical clustering of gene expression in different tissues. Data analyzed from Steuernagel et al (2018, BioRxiv). Read more about this study in our featured Application Note.
Application: Sensitive Detection of Rare Transcripts
Protocadherin genes, which exhibit stochastic transcriptional activation, are essential for establishing a neuronal surface identity code involved in neural circuit assembly during brain development. Researchers from Columbia University, Yale University, the Whitehead Institute for Biomedical Research, New York Genome Center and UCSF teamed up to address the mechanism behind the stochastic promoter choice of Pcdh (Canzio et al, 2019, Cell). However, the low level of expression of Pcdh genes provided significant cost and bioinformatic challenges to detect them in standard RNA-seq libraries. To overcome this challenge, the researchers used a myBaits Custom target capture kit as an efficient and cost-effective solution to enrich the Pcdh transcripts from RNA-Seq NGS libraries prior to sequencing. This solution allowed the researchers to achieve an over 45-fold enrichment of Pcdh transcripts using only 10% of the sequencing throughput of standard RNA-Seq, and thus dramatically increased sensitivity in detecting Pcdh expression. Learn more about this study in our featured Publication Note.
myBaits Enrichment of Rare Transcripts Enables High Coverage Sequencing. A high level of antisense RNA transcription of the Pcdhα alternate exons was revealed by myBaits Custom RNA-Seq capture with high confidence from increased coverage. Orange bar: myBaits for Pcdh α and γ clusters. Image based on Figure S1.E from Canzio et al (2019, Cell). Read more about this study in our featured Publication Note.
Application: Pathogen RNA-Seq Enrichment
It is often desirable to study the gene expression patterns of a particular pathogen while it is active in a host during an infection. However it is often impossible to physically separate host and pathogen RNA signals prior to total sample RNA-Seq, which then necessitates significant per-sample sequencing costs in order to sufficiently resolve the much lower signal of pathogen gene expression within the large ‘background’ of host RNA. Targeted enrichment via hybridization capture of just pathogen-derived transcripts, such as with myBaits Custom kits, is a comprehensive way to enrich the cDNA library just for pathogen gene signals of interest in order to significantly reduce NGS costs per-sample while still preserving the native transcript abundance signal.
In the following example, all genes from a fungal pathogen were enriched from total host+pathogen cDNA NGS libraries, with very high correlation of gene expression signals compared to non-enriched pathogen RNA-Seq libraries, as well as very high reproducibility of signal observed across 3 replicate experiments. This demonstrates that myBaits Custom target capture kits are a powerful tool for RNA-Seq studies of rare transcripts—such as pathogen gene expression in host background—that preserve critical abundance signals while significantly reducing per-sample NGS costs by orders of magnitude.
High correlation and reproducibility of myBaits pathogen RNA-Seq capture. Scatter plots of log2 reads counts from three replicates show a high correlation between enriched and non-enriched (shotgun) pathogen transcript read counts. The reads were obtained from a sample of host RNA spiked with 0.3% pathogen RNA.
Canzio, D. et al. 2019. Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice. Cell 177(3):P639-653.
Steuernagel, B. et al. 2018. Physical and transcriptional organisation of the bread wheat intracellular immune receptor repertoire. BioRxiv.