Oxford Nanopore Technologies have been initially developed to directly sequence the long molecules of DNA and RNA. The possibility of sequencing shorter molecules using nanopore is widely discussed in the field but remains questionable because of the higher error rate compared to the classical deep-sequencing approaches. Here we show the successful application of the MinION device to sequence tRNA molecules. The challenges of sequencing tRNAs are due its short length and folded structure, however, to overcome this we have improved the library preparation in order to compute the whole length of the tRNA. Initially, we sequenced a mixture of in vitro transcribed E. coli tRNAs and developed a bioinformatical pipeline to assign base-called reads to different tRNA species with a high degree of accuracy. Moving towards more complex samples containing native tRNAs, we found that modifications along the tRNA reduced the fidelity of called bases, so we developed an algorithm of tRNA classification based on raw-signal patterns. Comparing those patterns to unmodified in vitro transcribed tRNA signals allowed us not only to distinguish between different tRNA species, but also to detect modifications occurring in the native tRNAs. Our results show that nanopore-based approaches can be used to sequence tRNAs and classify them. This unveils a new area of the nanopore technology in application to short molecules, detecting the modifications and even predicting the potential ones, which are currently unknown, but may govern the structure, affect decoding or play a role in diseases.

Forensic DNA profiling uses short tandem repeat (STR) analysis for human identification purposes, i.e. to establish a link between biological evidence and an individual. This technique is currently limited to assessing the length of STR alleles via capillary electrophoresis and relies on the comparison to a reference DNA profile. The advent of DNA sequencing has revolutionised the field of forensic genetics. Alleles with the same length but a different sequence can be distinguished, providing additional discrimination between individuals which can greatly aid in DNA mixture interpretation. Rare sequence mutations can be identified to differentiate identical twins, who cannot be told apart using conventional DNA profiling. Using sequencing, scientists have also begun to harness intelligence-based information that a biological sample can provide which could be of use in an investigation. The analysis of single nucleotide polymorphisms (SNPs) offers new opportunities in the form of forensic DNA phenotyping and forensic epigenetics. Prediction of eye, hair and skin colour, as well as bio-geographic ancestry and chronological age estimations of an unknown individual are all now possible. The introduction of nanopore sequencing technology has the potential to transform the field of forensic genetics even further. The portability and real-time capability of the MinION could shift analysis out of the lab into the field, greatly reducing cost and turnaround time which are critical in an investigation. Research into the feasibility of this technology for forensic applications is currently underway. Sequencing has not only changed the field of forensic genetics, but also has changed the way biological evidence is approached and could be used in investigations which has had a wide-reaching effect in enforcement, legal, governmental and judicial fields. Although not routinely used in forensic casework at present, many forensic laboratories around the world are currently validating sequencing technologies with the expectation that this will be the biological evidence of the future.