Oxford Nanopore Launches cDNA Kits

By Bio-IT World Staff

February 5, 2019 Oxford Nanopore has launched new ‘109’ cDNA Kits for its real-time, scalable sequencing technology.   The new kits provide high throughput while generating complete sequences of full-length cDNA strands, with a low input option of just 1 ng PolyA+ RNA. 

Deeper insight into the dynamics of the transcriptome are critical in enabling researchers across a broad range of fields to understand how the stable DNA code of an organism is interpreted at a given moment in time, Oxford Nanopore believes. In healthcare, the identification of differentially spliced isoforms and fusion transcripts can inform disease diagnosis and treatment. RNA sequencing also provides a rapid, effective method of virus identification, and also has applications in environmental and agricultural science.  

Oxford Nanopore’s long-read cDNA sequencing solutions allow researchers to sequence millions of whole transcripts end-to-end in single reads. This enables users to simultaneously perform accurate isoform-level transcriptome characterization, and differential gene expression profiling without any of the compromises on cost, quantitation, computational or workflow complexity required to achieve this on other short or long read sequencing platforms.

The two kits now available are Direct cDNA (SQK-DCS109) and PCR-cDNA (SQK-PCS109) Sequencing Kits. In combination with upgrades to the Oxford Nanopore sequencing platforms and software, nanopore cDNA sequencing delivers effective enrichment for full-length transcripts (PCR-cDNA). In the case of the Direct cDNA kit, PCR is not necessary, removing bias, while the PCR-cDNA kit enables high throughput from low RNA input (just 1 ng PolyA+ RNA) and provides the option of starting from total RNA. The kits require less than 5 hours sample preparation time end-to-end, with little of this representing hands-on time. In addition to cDNA sequencing, Oxford Nanopore also offers direct RNA analysis kits, allowing users to sequence RNA without the need to convert to cDNA.

The new cDNA kits can be used on all Oxford Nanopore devices, the pocket-sized MinION, the benchtop GridION, and the high-throughput PromethION. This allows users to generate very high yields of transcriptomic data for high-resolution novel isoform discovery, identification of low copy number transcripts and de novo sequencing of entire transcriptomes to high coverage at any scale. Small, rapid devices such as Flongle are also ideal for targeted cDNA sequencing of specific transcripts.

Fly Embryo Use Case

Anthony Bayega and colleagues from McGill University used the new PCR-cDNA Sequencing Kit with a MinION to perform full-length cDNA sequencing of Poly-A+ RNA from mixed-sex olive fruit fly embryos. Bayega presented the research at a Nanopore Community Meeting, and Oxford Nanopore shared the use case when announcing the new products.

De novo transcriptome assembly identified 3,553 novel genes, 8,330 genes matching the predicted NCBI genes, and a total of 79,810 transcripts. Overall, a four-fold increase in transcriptome diversity compared to the NCBI predicted transcriptome was achieved. Furthermore, 38 genes incorrectly modelled by NCBI were corrected with this dataset.

“Nanopore cDNA Sequencing Kits have provided us with quick workflows, full-length quantitative transcripts, and eased our data analysis since complex statistical considerations that need to be taken for short-read data, where full-length resolution of genes is difficult to determine, are not necessary,” Bayega said in an Oxford Nanopore announcement of the products.

Absolute transcript numbers were determined by adding RNA standards (ERCC) during the cDNA synthesis stage; these were used to generate a standard curve for subsequent conversion of relative read counts to absolute counts of transcript abundance for each embryo. This quantitative approach was validated by qPCR measurement of gene expression. 

Using long-read sequences obtained from adult male and female olive fly heads, the isoform complexity of genes involved in sex determination was further explored. Understanding the mechanisms behind sex determination is important as it might reveal opportunities for genetic control. The researchers believe the work provides the first insight into the complexity of the olive fly embryo transcriptome and demonstrates how a greater understanding of sex determination has the potential to achieve population control of an agricultural pest of great economic impact.

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