Important Quality Update regarding NextSeq 2000 Runs

At NGI, we continuously monitor our sequencing output to ensure the highest standards of data quality. Over the past year, our team has been actively investigating intermittent data quality observations on our NextSeq 2000 platform. Below is a detailed overview of the issue, our interactions with Illumina, and the steps we are taking to resolve it.

Over the past year, NGI has occasionally observed elevated levels of N-bases / 0-intensity-cycles in NextSeq 2000 sequencing runs. We have not been able to link these occurrences to specific projects or sequencing setups. However, the frequency of the issue appears to increase with sequencing length, with Read 2 generally being more affected than Read 1. In some cases, identical library pools run with the same sequencing settings have produced substantially different levels of N bases.

NGI has informed Illumina about this issue and in some cases, Illumina has replaced the flowcell. However, for other runs that NGI considered problematic, Illumina assessed the flowcell as performing within specification. It is important to note that N-base levels themselves are not part of Illumina’s specifications. Instead, flowcell performance is assessed using metrics such as the mapping rate of the PhiX control. According to Illumina’s specifications, a run may therefore meet the required performance criteria even if entire cycles contain a high proportion of N calls.

Based on Illumina’s recommendations, NGI has evaluated different loading conditions, including adjustments to PhiX spike-in levels and loading amounts. While these changes have resulted in minor improvements in some cases, they have not consistently eliminated the occurrence of elevated N-base calls.

NGI is committed to providing sequencing data of the highest possible quality and yield to our users. As increasing the PhiX fraction did not sufficiently address the issue and also reduced the number of usable library reads, NGI continued to discuss the matter with Illumina. As a further step in the investigation, Illumina has provided NGI with a loaner NextSeq 2000 instrument, which we are currently using to investigate if the issue is instrument related.

Last Updated: 12th October 2026