Direct poly(A) RNA nanopore sequencing on the freshwater duck mussel Anodonta anatina following exposure to copper : A pilot study

Sammanfattning: Aquatic ecotoxicology is the study of toxic chemicals and its effects on aquatic biological systems with the aim of minimising threats to human health and ensure self-sustained ecosystems. Freshwater bivalves are excellent sentinels for use in ecotoxilogical research due to their filter feeding properties, stationary lifestyle and inability to regulate body temperature. This project aimed to assess the feasibility and use of nanopore sequencing, a real-time single-molecule sequencing technology in comparative expression analysis by sequencing transcriptomic RNA from the freshwater mussel Anodonta anatina following exposure to copper. RNAs were extracted from 80 mg hepatopancreas tissue, followed by poly(A) RNA selection. Furthermore, the poly(A) RNA was used to construct a nanopore sequencing library. Sequencing a total amount of 560 ng poly(A) RNA over the course of two separate runs generated 239,448 reads, in which 75% of the reads were obtained during the first run (control) and 25% of the reads were obtained during the second run (case). The median read lengths ranged between 534-650 nucleotides, with a base call accuracy <90%. Due to the big differences in sequence data output between the two sequencing runs, the data was ineligible for comparative analysis. The findings conclude that nanopore sequencing is capable of generating longer read lengths when compared to other sequencing platforms. However, the technology is error-prone in terms of accurate base call identifications and relies on other platforms for error corrections. Future advances include de novo transcriptome assembly for efficient use of Anodonta anatina as a bioindicator in aquatic ecotoxicology.