Accurate gene consensus at low nanopore coverage.

Area of Science:

• Genomics
• Bioinformatics
• Molecular Biology

Background:

• Nanopore sequencing offers high throughput but suffers from high error rates.
• High error rates in nanopore reads obscure low-frequency mutations in amplicon libraries.
• Existing consensus calling methods reduce errors but decrease throughput by requiring many reads.

Purpose of the Study:

• To introduce SINGLe (SNPs In Nanopore reads of Gene Libraries), an innovative error correction method.
• To reduce sequencing noise in nanopore reads of amplicons with point variations.
• To improve the accuracy of variant detection in amplicon sequencing.

Main Methods:

• SINGLe leverages the similarity of reads in an amplicon library to a wild-type sequence.
• It characterizes position-specific systematic error patterns of the nanopore basecaller.
• The method reweights nucleotide confidence based on wild-type comparison and incorporates this into consensus calling.

Main Results:

• SINGLe was tested on a mutagenic KlenTaq polymerase gene library with sub-sequencing noise mutation rates.
• The method effectively compensates for systematic errors inherent in nanopore basecalling.
• SINGLe achieved accurate consensus sequences with as few as 5 reads per variant.

Conclusions:

• SINGLe outperforms existing methods in accuracy and efficiency for nanopore amplicon sequencing.
• The method enables reliable detection of low-frequency mutations previously masked by sequencing noise.
• SINGLe significantly reduces the number of reads required for accurate variant identification.