Cross-platform comparison of salus pro and illumina NextSeq 550Dx for clinical targeted sequencing

High-throughput sequencing serves as an indispensable tool for precise molecular diagnosis in clinical practice. While platforms such as Illumina NextSeq 550Dx are widely used in clinical diagnostics and assay development, their high costs and reliance on proprietary reagents restrict widespread adoption. The Salus Pro is an emerging sequencing platform that offers operational flexibility and broad library compatibility; however, its clinical performance requires systematic validation. This study aimed to evaluate the performance of the Salus Pro through a head-to-head comparison with the clinically established Illumina NextSeq 550Dx. Using a parallel testing design, cholangiocarcinoma patient derived 242 DNA and 274 RNA libraries originally sequenced on the NextSeq 550Dx were re-sequenced on the Salus Pro. In-depth bioinformatic analysis was subsequently performed to compare key performance metrics, including sequencing quality (Q30), read alignment rate, on-target rate, sequencing depth, and variant detection concordance. The Salus Pro exceeded its own design specifications and exhibited consistent performance across batches. Q30 score were above 93%, sequencing output exceeded chip specification by 20%-60%, and the sample index recognition rate was above 97%. In the head-to-head comparison, the Salus Pro showed higher Q30 scores than the NextSeq 550Dx, with a mean paired difference of 4.15% (95% CI: 3.90%–4.40%) for DNA, and 4.72% (95% CI: 4.51%–4.94%) for RNA. The on-target rate and sequencing depth in DNA targets were also higher on the Salus Pro, with mean paired differences of 4.58% (95% CI: 4.35%–4.80%) and 44.56 × (95% CI: 23.77×-65.35×), respectively. At the clinical endpoint, the two platforms showed concordance in variant detection, with high correlations for both DNA variant allele frequencies (Spearman r = 0.9501, p-value < 1e-20) and RNA fusion supporting reads (Spearman r = 0.8536, p-value = 4.45e-20). Variant-level concordance was further assessed using positive and negative percent agreement for representative hotspot variants. For hereditary disease samples, the Salus Pro also showed consistent performance, with an average alignment rate of 100%, an average paired-end rate 98.86%, and an average Q30 of 94.61%. In summary, the Salus Pro met all quality control requirements and exceeded its design specifications in key parameters. Within the evaluated disease types and targeted panel context, it demonstrated comparable performance to the Illumina NextSeq 550Dx, with improvements in selected sequencing metrics. These findings provide experimental evidence supporting further evaluation of the Salus Pro in clinical genetic testing.