METHOD TO ANALYZE tRNA USING DIRECT SEQUENCING

A method to quantify tRNA abundance and tRNA modifications that comprises the following steps:

• a) contacting an RNA sample containing tRNA, in the presence of an agent with ligating activity, with:
• a pre-annealed splinted double-stranded oligonucleotide comprising:
• a first splinted oligonucleotide, comprising a second splinted oligonucleotide hybridization region and a tRNA hybridization region, said tRNA hybridization region being located on its 3′ end,
• a second splinted oligonucleotide, comprising a first splinted oligonucleotide hybridization region, a second adapter DNA oligonucleotide hybridization region, such that at the end of the step, the first splinted oligonucleotide is adjacent to the 5′ end of the tRNA and complementary annealed to both the 3′ end of the tRNA by the tRNA hybridization region and to the second splinted oligonucleotide by the second splinted oligonucleotide hybridization region,
• b) contacting the product of step a) in the presence of an agent with ligating activity with a pre-annealed adapter DNA double-stranded oligonucleotide comprising:
• a first adapter DNA oligonucleotide, comprising a second DNA adapter oligonucleotide hybridization region, and
• a second adapter DNA oligonucleotide, comprising a first DNA adapter oligonucleotide hybridization region and a second splinted oligonucleotide hybridization region, being said second splinted oligonucleotide hybridization region complementary to the second adapter DNA oligonucleotide hybridization region of the second splinted oligonucleotide,
• such that at the end of the step, the first adapter DNA oligonucleotide is adjacent to the 3′ end of the terminal region of the second splinted RNA oligonucleotide, and the second DNA oligonucleotide is complementary annealed to both the second splinted RNA oligonucleotide and the first adapter DNA oligonucleotide,
• c) performing reverse transcription to linearize the product of step b) to obtain a library and
• d) carrying out nanopore direct sequencing with the library of step c), to obtain the abundance of tRNA and its modifications in said RNA sample.