Rebekah and Abbie were awarded flexible funding from the MRC to attend a two-day training course down at the Oxford Nanopore Headquarters. The training course specifically focused on direct RNA sequencing, with hands on elements and a comprehensive data analysis session which introduced ONP recommended bioinformatics tools for sequence analysis.
Over the last decade, improvements in next generation sequencing have transformed the field of genomics and transcriptomics. The ability to rapidly sequence whole genomes, specific genomic regions and transcripts of interest is paving the way in the development of new insights into diagnostics and medical care, risk management, metagenomics, antimicrobial resistance, evolutionary biology and crop breeding. Although we are presently living in what has been proclaimed as the area of the sequencing revolution; the massive up-front costs associated with sequencing prohibit many labs from utilising these technologies in house. This inaccessibility has driven the trend of outsourcing; especially when it comes to whole genome sequencing. One company that is aiming to cause a paradigm shift when it comes to genetic analysis, is Oxford Nanopore. Oxford Nanopore’s ethos is that sequencing is an essential capability for modern research laboratories and thus aim to widen access to in house DNA/RNA technology.
MinION is a handheld sequencing tool developed by the UK company Oxford Nanopore. MinION is portable, real-time, and comes with a relatively low-price-tag (the starter pack costs $1,000). The device contains an array of micro scaffolds that support a polymer membrane containing thousands of embedded nanopores. Once loaded and connected to a power source, an ionic current pass through each nanopore generating a voltage difference across the membrane. During sample preparation “adapter” proteins are ligated to the ends of genomic DNA or cDNA strands. The adaptor proteins direct the strands to the membrane where they pass through the pore in a unidirectional flow. As the nucleic acids are transported through a pore, each base disrupts the current in a unique fashion; allowing the sequence to be obtained. By 2014, the MinION, a hand-held portable sequencing device was introduced to an early access scheme, and in 2015 it became commercially available.
During library preparation, genetic material within the sample is not intentionally fragmented, enabling the MinION to generate sequence reads which are substantially longer than other comparative platforms. Traditional sequencing methods rely on the fragmentation and in sillico reassembly of DNA/RNA sequences. This fingerprinting approach can sometimes yield poorly constructed, low quality assemblies, especially when it comes to assembling complex genomic regions and highly identical sequences which can lead to missing information and genomic reduction. Longer read lengths allow for overlapping so that the sequence can be unambiguously reconstructed. Long read lengths also enable the identification of epigenetic modifications and transcript isoforms. Apart from human applications, other organisms such as plants have highly repetitive sequences making it difficult to obtain a complete genome using short read methods. Aside from longer read lengths, MinION also has the advantage of being able to directly sequence native RNA and DNA enabling the detection of individual nucleotide modifications.
As mentioned previously, Nanopore sequencing can be used to perform direct RNA sequencing, giving real-time sequence data without the need for reverse transcription or amplification steps that are known to introduce biases. Full length, strand-specific RNA sequences can be generated, and the method also permits the detection of nucleotide modifications. In order to conduct direct sequencing, first, an Oligo (B) complementary to the polyA tail of mRNA or to a target of interest (10bp) is annealed to the strand of interest. Then, a second Oligo (A) binds to oligo (B). The binding of the second Oligo allows the strand of interest being directed towards the nanopore to be sequenced.
As the RNA passes through the pore, the ionic current changes and is recorded as a “squiggle” in a fast5 format. Basecalling algorithms convert this squiggle data into fastqs. Which can then be taken into bioinformatic analysis. Basecalling software algorithms are always improving, and rebasecalling can improve the quality of your reads. Fast5 files, when directly sequencing RNA, can reveal information about base modifications.
This training was invaluable and has informed both Rebekah and Abbie’s PhD projects immensely.