Chasing Sequencing Perfection: Marching Toward Higher Accuracy and Lower Costs

Abstract Next-generation sequencing (NGS), represented by Illumina platforms, has been an essential cornerstone of basic and applied research. However, the sequencing error rate of 1 per 1000 base pairs (10−3) represents a serious hurdle for research areas focusing on rare mutations, such as somatic mosaicism or microbe heterogeneity. By examining the high-fidelity sequencing methods developed in the past decade, we summarized three major factors underlying errors and the corresponding 12 strategies mitigating these errors. We then proposed a novel framework to classify 11 preexisting representative methods according to the corresponding combinatory strategies and identified 3 trends that emerged during methodological developments. We further extended this analysis to 8 long-read sequencing methods, emphasizing error reduction strategies. Finally, we suggest 2 promising future directions that could achieve comparable or even higher accuracy with lower costs in both NGS and long-read sequencing.