Sequencing is a key tool in a biologist’s arsenal, but with new technology popping up in the midst of ever-decreasing costs, it can be hard to figure out which is the best method to use. In a recent webinar, Claire Bailey Hartmann (Director of Bauer Core Facilities, Harvard University) gave us a rundown of how to choose a platform for your sequencing experiments. In this article, we summarise the key points you should consider when making this decision.
Watch the full Sequencing 101 webinar here.
Is sequencing right for you?
This might not be the first question you were expecting – but it’s an important one. Sequencing can be a crucial part of many experiments, but sometimes there will be cheaper, more efficient or even more sensitive tools you can use to answer your question. These methods include digital PCR and imaging, among others. Before starting your experiment, consider whether you need to perform any sequencing.
DNA or RNA?
If sequencing is the right tool for you, what happens next depends on whether you are going to be sequencing DNA or RNA. Both serve their purpose and can be effective ways to answer different questions – a DNA sequence can provide static insights into the genome, whilst RNA sequencing can provide a more dynamic view of gene expression.
DNA sequencing
You’ve chosen to do DNA sequencing – but what are you looking to do?
De novo assembly
Previously, de novo builds could be complex, and long-read data would need to be complemented by short-reads. Recent advances have made long-read only assemblies possible, making de novo builds more accessible. As before, the ideal technology varies depending on your question and research needs. PacBio, for example, provides high quality and highly accurate reads compared to Nanopore sequencing. Conversely, Nanopore has an advantage when it comes to coverage, can be less expensive for large genomes, and is more tolerant to DNA fragments of different sizes, meaning it can be sequence larger elements of the genome in one read.
Resequencing
Resequencing can be performed when a reference genome for the organism already exists and is typically used to look for variants in a sample against this reference. In this case, high accuracy is vital to differentiate between sequencing errors and genuine variation. The need for higher accuracy is directly related to how rare the variant is. Illumina tools are commonly used for resequencing, but some newer instruments, for example from PacBio or Ultima, can also be effective for resequencing.
DNA modifications
Historically, bisulphite sequencing was used to identify modifications to the DNA. Illumina was once the go-to for this purpose. However, PacBio and Nanopore sequencing instruments are both now capable of directly detecting modifications. PacBio achieves this by analysing the time it takes for a nucleotide to be added to a sequence, which can indicate a modification. On the other hand, Nanopore achieves this by assessing the disruption of the charge as the DNA crosses the membrane. Nanopore tools can, in theory, be used to detect any kind of modification, but training a data set for this purpose can be a difficult and time-consuming task.
RNA sequencing
RNA sequencing be used to sequence bulk samples, single-cells or to provide spatial context.
There are several widely used single-cell RNA sequencing approaches. Some of these do not require instrumentation, making them accessible to more researchers. The choice of which to use again depends on your question and, notably, the number of cells you wish to sequence.
There are also a number of tools available for bulk RNA sequencing. The choice of which to use can depend on whether a suitable reference genome is available. If not, Claire recommends using long-read sequencing to more easily perform de novo assembly. Which long-read sequencing tool you would use depends on whether you are prioritising accuracy or cost – PacBio and Nanopore perform well in these categories, respectively. If a reference genome is available, Claire recommends short-read technology for gene level analysis and long-read for isoform level analysis.
As exciting as spatial context may be, gathering this data can be expensive and challenging. Therefore, Claire suggests only performing spatial transcriptomics if your question truly requires it. If so, there are a number of non-sequencing techniques that can be used to provide this context, such as imaging.
Want to know which tools Claire recommends? Watch the full webinar here.
Top tips
Ultimately, Claire’s key take home message is that if you are in doubt, ask a core scientist for advice. The right tool for you depends on your question, and you should take the time to determine the optimal solution.
