Methods for identifying and using small RNA predictors
The invention provides a method for identifying or detecting small RNA (sRNA) predictors of a disease or a condition. The method comprises identifying one or more sRNA sequences that are present in one or more samples of an experimental cohort, and which are not present across a comparator cohort; and optionally identifying one or more sRNA sequences that are present in one or more samples of a comparator cohort, and which are not present across an experimental cohort. In contrast to identifying dysregulated non-coding RNAs (such as miRs that are up- or down-regulated), the invention identifies sRNAs that are binary predictors, that is, present in one cohort (e.g., an experimental cohort) and not another (e.g., a comparator cohort). Further, by quantifying reads for individual sequences (e.g., iso-miRs), without consolidating reads to annotated reference sequences, the invention unlocks the diagnostic utility of miRs and other sRNAs.
1. A method for identifying small RNA (sRNA) predictors for detection of cancer, comprising:
providing sRNA sequencing data from biological samples of an experimental cohort and a comparator cohort, the sRNA sequencing data being generated by a sequencing-by-synthesis sequencing platform; wherein the samples of the experimental cohort and the samples of the comparator cohort are defined by the presence or absence, respectively, of a cancer;
wherein the sRNA sequencing data comprises sequence reads having adaptor sequences at 3′-ends, and sRNA sequences are defined from the sequence reads by deletion of the adaptor sequences so as to identify variations at the 3′- and 5′-ends of the sRNA sequences, the sRNA sequences being defined without consolidating sequence reads to a reference sequence;
selecting sRNA sequences that are present in at least 10% of the biological samples in the experimental cohort and which are not present in any samples of the comparator cohort, thereby identifying positive sRNA predictors;
detecting the presence and absence of the selected positive sRNA predictors in RNA extracted from samples of a validation cohort using a quantitative or qualitative PCR assay that is specific for the sRNA predictors, thereby identifying sRNA predictors for detection of the cancer.
2. The method of claim 1 , wherein the biological samples are solid tissue, biological fluid, or cultured cells.
3. The method of claim 2 , wherein the biological samples are biological fluid samples selected from blood, serum, plasma, urine, saliva, or cerebrospinal fluid.
4. The method of claim 1 , wherein the experimental cohort and the comparator cohort each have at least 100 samples.
5. The method of claim 1 , wherein the positive sRNA predictors are identified by quantifying the number of reads for each unique sRNA sequence in each sample of the experimental cohort.
6. The method of claim 5 , wherein the sequence reads from the experimental cohort and the comparator cohort are compiled, and compared; and where sequence reads that are in both cohorts are discarded, and sequence reads that are unique to the experimental cohort are candidate sRNA predictors.
7. The method of claim 6 , wherein an output file annotates the unique sequences, and annotates the count of the unique reads for each sample or group of samples in the experimental and comparator cohorts.
8. The method of claim 7 , wherein sRNA predictors are selected that have a sequence read count of at least 5 in the majority of samples that are positive for the predictor.
9. The method of claim 8 , wherein positive sRNA predictors are selected that are present in at least 20% of samples in the experimental cohort.
10. The method of claim 9 , wherein from 2 to 50 sRNA predictors are selected for inclusion in an sRNA predictor panel.
11. A method for detecting cancer in a subject, comprising: providing a biological sample from the subject, and identifying the presence or absence of the sRNA predictors identified according to the method of claim 1 , wherein the sRNA predictors are detected using a PCR assay employing a stem loop primer and a fluorescently-labeled probe, wherein the presence of one or more sRNA predictors in the sample is indicative of the presence of cancer, and wherein one or more of the sRNA predictors are isomiRs.
12. The method of claim 1 , wherein at least one identified sRNA predictor is an isomiR.
13. The method of claim 1 , wherein the PCR assay employs a stem loop primer and a fluorescently-labeled probe.
14. The method of claim 1 , wherein the experimental cohort and the comparator cohort have at least 25 samples.
15. The method of claim 1 , wherein the experimental cohort and the comparator cohort have at least 50 samples.
16. The method of claim 1 , wherein the cancer is a carcinoma, sarcoma, or lymphoma.
17. The method of claim 1 , wherein the cancer is lung, skin, breast, ovary, intestine, pancreas, bone, or brain.
18. The method of claim 1 , wherein the cancer is breast cancer.
19. The method of claim 11 , wherein the cancer is lung, skin, breast, ovary, intestine, pancreas, bone, or brain.
20. The method of claim 11 , wherein the cancer is breast cancer.
21. The method of claim 11 , wherein the presence or absence of at least 5 sRNA predictors is tested in the sample.
22. The method of claim 11 , wherein the sample is blood, serum, or plasma.