Low-yield respiratory sequencing in pediatric upper respiratory specimens: a case series and reporting framework

Clinical interpretation of respiratory sequencing results is difficult when analytical support is sparse or when sequencing findings do not align with routine laboratory reports. We expanded an ultra-low-yield index case into a retrospective descriptive pediatric case series to characterize recurrent interpretive scenarios and support a pragmatic laboratory reporting framework. We retrospectively reviewed archived upper respiratory specimens from pediatric patients with respiratory symptoms who had undergone both routine respiratory testing and sequencing-based pathogen analysis. Clinical features, routine-test interpretation, sequencing metrics, top reported hits, result-return timing, management review, and short-term outcomes were abstracted from retrievable records. Ten children aged 6-10 years were included. Routine testing was classified as influenza-positive in 8 cases and negative in 2 cases. Retained pathogen-associated contigs were sparse (median: 12.5; range: 5-17), and mapped read support was low (median: 408.5 read pairs; range: 384-453). Top low-support hits included rhinovirus/rhinovirus B in 6 cases, respiratory syncytial virus in 2 cases, and Mycoplasma-related hits in 2 cases. The Mycoplasma-related findings were interpreted cautiously because limited report-level sequencing evidence and the absence of orthogonal confirmation, paired serology, lower-respiratory specimen confirmation, or specimen-matched negative-control review prevented confident distinction between active infection, carriage or colonization, transient detection, coinfection of uncertain relevance, and contamination. No case had documented orthogonal confirmation or a specimen-matched negative control. Provider-level clarification indicated the use of batch-level negative controls, the absence of respiratory pathogen-related background reads, contamination-aware filtering, and manual review, although raw batch-level quality-control (QC) reports were not independently retrievable. Low-yield respiratory sequencing results in this small, purposively selected pediatric series were best understood as analytically limited signals requiring cautious interpretation. Accordingly, these low-support detections should be treated as hypothesis-generating observations rather than disease-defining findings. The proposed framework should be interpreted as a preliminary reporting aid for structured interpretation, not as a validated diagnostic algorithm.