We performed sncRNA-seq for the PBMC samples collected from 59 human subjects in the discovery cohort, including 13 healthy controls, 10 pulmonary TB patients, and 36 lung cancer patients (Additional file 2: Table S1). The raw sequencing data were processed by our newly developed computational framework, SPORTS1.0, which was designed to optimize the annotation and quantification of non-canonical sncRNAs (i.e., tsRNAs, rsRNAs, and ysRNAs) in addition to miRNAs [2]. In total, 6673 tsRNA species, 20,172 rsRNA species, 1238 ysRNA species, and 973 miRNA species were identified in human PBMCs (Additional file 1: Fig. S2). We investigated the co-expression pattern of tsRNAs across the PBMC samples in the discovery cohort by grouping tsRNA species into subcategories according to their parent tRNA types. We found that the expression of the tsRNAs derived from the tRNAs of alanine (tsRNA-Ala), asparagine (tsRNA-Asn), leucine (tsRNA-Leu), lysine (tsRNA-Lys), and tyrosine (tsRNA-Tyr) was strongly and positively correlated with that of each other (Spearman’s rank correlation test: ρ > 0.700 and P < 10− 9) (Fig. 1a), suggesting shared biogenesis pathways among these tsRNAs. Interestingly, tsRNA-Ala, tsRNA-Asn, tsRNA-Leu, tsRNA-Lys, and tsRNA-Tyr were the only five tsRNA groups that were upregulated in the lung cancer patients relative to the controls (adjusted P < 0.05) (Fig. 1b). We further found that the expression of these five tsRNA groups was also significantly higher in the lung cancer patients than in the pulmonary TB subjects (P < 0.05) (Fig. 1b). We next grouped rsRNA and ysRNA species into subcategories according to their parent rRNA/YRNA types and found that the rsRNAs derived from rRNA-5S (rsRNA-5S) were significantly upregulated in the lung cancer patients relative to the controls, while the ysRNAs originating from YRNA-RNY1 (ysRNA-RNY1) were downregulated in the lung cancer patients compared with the controls (adjusted P < 0.05) (Fig. 1b). More interestingly, the expression of rsRNA-5S and ysRNA-RNY1 showed a completely inverse pattern in the pulmonary TB patients: rsRNA-5S was significantly downregulated in the TB patients relative to the controls, while ysRNA-RNY1 was upregulated in the TB patients compared with the controls (P < 0.05) (Fig. 1b). We further mapped the individual tsRNA-Ala, tsRNA-Asn, tsRNA-Leu, tsRNA-Lys, tsRNA-Tyr, rsRNA-5S, and ysRNA-RNY1 species to the corresponding parent RNAs and identified a nonrandom fragmentation pattern (Fig. 1c-d and Additional file 1: Fig. S3), suggesting highly regulated biogenesis of these sncRNAs. In addition, we investigated the association of these non-canonical sncRNA expression with cancer stage, histological type, lymph node status, metastasis status, and smoking history, but no significant difference was observed (Additional file 1: Fig. S4-S8).