Estimated Nucleotide Reconstruction Quality Symbols of Basecalling Tools for Oxford Nanopore Sequencing

Currently, one of the fastest-growing DNA sequencing technologies is nanopore sequencing. One of the key stages involved in processing sequencer data is the basecalling process, where the input sequence of currents measured on the nanopores of the sequencer reproduces the DNA sequences, called DNA reads. Many of the applications dedicated to basecalling, together with the DNA sequence, provide the estimated quality of the reconstruction of a given nucleotide (quality symbols are contained on every fourth line of the FASTQ file; each nucleotide in the FASTQ file corresponds to exactly one estimated nucleotide reconstruction quality symbol). Herein, we compare the estimated nucleotide reconstruction quality symbols (signs from every fourth line of the FASTQ file) reported by other basecallers. The conducted experiments consisted of basecalling the same raw datasets from the nanopore device by other basecallers and comparing the provided quality symbols, denoting the estimated quality of the nucleotide reconstruction. The results show that the estimated quality reported by different basecallers may vary, depending on the tool used, particularly in terms of range and distribution. Moreover, we mapped basecalled DNA reads to reference genomes and calculated matched and mismatched rates for groups of nucleotides with the same quality symbol. Finally, the presented paper shows that the estimated nucleotide reconstruction quality reported in the basecalling process is not used in any investigated tool for processing nanopore DNA reads.

Automated summary

Nanopore sequencing is a rapidly growing DNA sequencing technology. The basecalling process, which reproduces DNA sequences from the measured current sequence on the nanopores of the sequencer, is a key stage in processing sequencer data. This study compares the estimated quality of nucleotide reconstruction reported by different basecallers and investigates its usage in processing nanopore DNA reads. The results demonstrate variations in estimated quality and the lack of utilization of this information in existing tools.