Sequencing error profiles of Illumina sequencing instruments

Sequencing technology has achieved great advances in the past decade. Studies have previously shown the quality of specific instruments in controlled conditions. Here, we developed a method able to retroactively determine the error rate of most public sequencing datasets. To do this, we utilized the overlaps between reads that are a feature of many sequencing libraries. With this method, we surveyed 1943 different datasets from seven different sequencing instruments produced by Illumina. We show that among public datasets, the more expensive platforms like HiSeq and NovaSeq have a lower error rate and less variation. But we also discovered that there is great variation within each platform, with the accuracy of a sequencing experiment depending greatly on the experimenter. We show the importance of sequence context, especially the phenomenon where preceding bases bias the following bases toward the same identity. We also show the difference in patterns of sequence bias between instruments. Contrary to expectations based on the underlying chemistry, HiSeq X Ten and NovaSeq 6000 share notable exceptions to the preceding-base bias. Our results demonstrate the importance of the specific circumstances of every sequencing experiment, and the importance of evaluating the quality of each one.

Automated summary

This study developed a method to retrospectively determine the error rate of public sequencing datasets, finding that expensive platforms have lower error rates and less variation, but there is significant variation within each platform, with experiment accuracy depending greatly on the experimenter. The importance of sequence context and differences in sequence bias patterns between instruments were also highlighted.