Written by Vered Smith, Science Writer
A paper in Molecular Biology Reports has recently reviewed the latest RNA sequencing technologies, and how they can be applied to help treat human diseases.
RNA sequencing (RNA-seq) gives an insight into the genomic processes that leading to disease. This field has been revolutionised by next-generation sequencing of transcriptomes. Investigating differential gene expression (DGE) to compare normal cells/tissues with diseased cells/tissues can help to identify the cause of different diseases. This knowledge can then be applied to improving diagnosis and the development of new treatments.
RNA Sequencing Techniques
There are several types of RNA sequencing techniques, each with their own advantages and disadvantages.
Short-read cDNA sequencing involves RNA extraction, mRNA enrichment and fragmentation, cDNA synthesis and fragmentation, cDNA amplification, sequencing, and data analysis The base pair banding of mRNA fragments needs to be 150 – 200 base pairs long for library purification and preparation, so the prepared cDNA is usually 200 – 400 base pairs long. This method is therefore limited to shorter transcripts, and is inaccurate at analysing multi-mapped reads.
Long-read cDNA sequencing has solved this shortcoming. It can process the full length of long RNAs. Single-Molecule Real-Time (SMRT) technology from Pacific Biosciences and nanopore sequencing technology from Oxford Nanopore Technologies can carry out this process. The disadvantage is that it takes longer, so further studies are needed to make the process more time-efficient.
Long-read direct RNA sequencing (DRS) can be performed on Oxford Nanopore’s sequencing technology. The main steps include ligating the duplex adaptor to the polyA tail of RNA, performing reverse-transcription, attaching the motor protein-attached sequencing adaptor, and preparing the library. It does not copy the RNA into cDNA, so reduces the possibility of errors occurring during that step. This technique can identify RNA base modifications, which is extremely useful for investigating epigenetics. However, the fragmentation of the input read is still a challenge.
Applications of RNA Sequencing in Disease
There are many examples of the use of RNA-seq to improve the treatment of cancer. Analysing short RNA fragments of ACTB and HER2 detected the intratumour heterogeneity of breast cancer cells at a molecular level. In both lung adenocarcinoma and chondrosarcoma, the identification of mutations in MET and isocitrate dehydrogenase 1 (IDH1) has opened the possibility of designing therapy targeted against them. RNA-seq can identify the tumour mutational burden, which is a potential immune checkpoint biomarker, and can help with a more accurate cancer prognosis. In head and neck cancer and oligodendroglioma, RNA-seq detected copy number variations of benign and malignant cells to determine the differences between them. RNA-seq can be used as a tool in diagnosing blood-based sarcoma.
RNA-seq has also been applied to improving our understanding of rare diseases. Some successful examples include: the measurement of allele-specific expression in idiopathic cardiomyopathy, the identification of the causal gene of enoyl CoA reductase protein-associated neurodegeneration (MEPAN), the identification of variants in regulatory upstream regions of genes in congenital muscular dystrophy, and the detection of a splice-affecting variant to diagnose collagen VI dystrophy.
Information about viral diseases can be provided by RNA-seq, and DRS has sequenced full-length virus transcriptomes. The HCoV-229E virus, part of the coronavirus family, contains the largest known RNA genome. This has been sequenced using nanopore technology, and the data can be used to analyse the cause of coronavirus resistance to current treatments.
Future Perspectives
With ongoing advancements in RNA-seq technology, its use as part of standard diagnostic tools in cancer and rare disease is expected in the future. Furthermore, it has the potential to enable the development of targeted therapies that are more effective and have fewer side effects.
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