Next-generation sequencing (NGS) is the modern, second revolution and the spearhead of an ever-accelerating field. The NGS technology performs innovative research in a variety of different areas, such as BioPharm, Population genetics/genomics, Agriculture, Forensics, Water testing, Complex and Infectious disease research and etc. RNA sequencing (RNA-seq) must be the most mature one of NGS. Here is what you need to know to begin using this tool.
Choosing the right RNA-Seq depends on your research question and you first need to understand what type of RNA you are studying. In eukaryotic total RNA, there are various types of RNA molecules, such as:
- ribosomal RNA (rRNA) – 80-90% of total eukaryotic RNA is in fact rRNA
- transfer RNA (tRNA)
- messenger RNA (mRNA) – only 3-7% total eukaryotic RNA represents the protein coding mRNA
- non-coding RNA (ncRNA), such as for instance:
- long non-coding RNA (lncRNA)
- circulatory RNA (circRNA)
- micro RNA (miRNA)
So how can you know what type of RNA you wish to study? To do that, you need to consider your research objectives: for instance, some projects are interested ‘only’ in comparing transcriptomic profiles across samples in different experimental conditions (e.g Treatment vs. Control), or between different tissue types, to identify tissue-specific gene expression. Other research groups might be more interested in analyzing a shift in cellular transcriptomic footprints across time to determine the time-scale gene expression patterns.
When it comes to transcript profiling, there are three major questions you would wish to answer. One of them is Annotation – a step where you assign a function to your RNA molecules. An important aspect to keep in mind is that when analyzing mRNA-Seq data originating from eukaryotic organisms, various isoforms are formed in a process called alternative splicing, and you might want to consider sequencing the different splice variants as well. The second thing you want to do is the Quantification of target RNA molecules – crucial for the differential gene expression analysis, where you aim to determine which genes are upregulated/downregulated across your samples. Lastly, one can also perform a Target prediction/network study – particularly important when working with non-coding RNA molecules, where you aim to investigate the potential interaction between various RNA molecules, and what are the potential targets of these ncRNAs.
Now that we know all this, you might want to ask yourself what sequencing technologies are available? As a sequence service provider, at Novogene we have the most advanced sequencing platforms to cater to diverse sequencing needs. One of these platforms is the NovaSeq6000 short-read sequencing machine, the most commonly used NGS sequencer which can sequence all types of RNA molecules (up to 500 bp). We also use long-read sequencing Nanopore and PacBio platforms, capable of sequencing up to 10kB in length. However, these platforms can only be used to sequence eukaryotic mRNA molecules containing a polyA tail.
While opting for long-read sequencing approaches may intuitively seem like a better option since full-length transcripts are obtained (meaning that no short-read assembly is required), and PCR bias and input RNA fragmentation are avoided, it is important to note that there are also some disadvantages that need to be considered before deciding to deploy this method. These cons include high error rates, extremely high sample requirements, and the fact that quantification still needs to be done with short-read NGS data.