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, comprising:
identifying one or more sRNA sequences that are present in one or more biological samples in an experimental cohort, and which are not present in samples of a comparator cohort, thereby identifying a positive sRNA predictor.
2 . The method of claim 1 , further comprising identifying one or more sRNA sequences that are present in one or more samples in a comparator cohort, and which are not present in samples of an experimental cohort, thereby identifying a negative sRNA predictor.
3 . The method of claim 1 or 2 , wherein the one or more sRNA sequences are identified using RNA sequencing data for the experimental and comparator cohorts.
4 . The method of any one of claims 1 to 3 further comprising, detecting the sRNA predictor(s) in independent experimental and/or comparator samples.
5 . The method of claim 4 , wherein the sRNA predictor(s) are detected in an independent cohort using a quantitative or qualitative PCR assay.
6 . The method of any one of claims 1 to 5 , wherein the biological samples are solid tissue, biological fluid, or cultured cells.
7 . The method of claim 6 , wherein the biological sample is a sample from animal, plant, or microbe.
8 . The method of claim 6 , wherein the biological samples are biological fluid samples selected from blood, serum, plasma, urine, saliva, or cerebrospinal fluid.
9 . The method of any one of claims 1 to 8 , wherein the experimental cohort and the comparator cohort each have at least 10 samples.
10 . The method of claim 9 , wherein the experimental cohort and the comparator cohort each have at least 100 samples.
11 . The method of any one of claims 1 to 10 , wherein the experimental cohort comprises samples from patients diagnosed as having a neurodegenerative disease, a cardiovascular disease, an inflammatory or immunological disease, or a cancer.
12 . The method of claim 11 , wherein the patients in the experimental cohort are diagnosed as having a neurodegenerative disease selected from Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease, or Multiple Sclerosis.
13 . The method of any one of claims 1 to 12 , wherein the positive sRNA predictor(s) are identified by quantifying the number of reads for each unique sRNA sequence in each sample of the experimental cohort; and the negative sRNA predictor(s) are identified by quantifying the number of reads for each unique sRNA sequence in each sample of the comparator cohort.
14 . The method of claim 13 , wherein a user-defined 3′ sequencing adaptor is trimmed from the sequence reads.
15 . The method of claim 14 , wherein the following regular expressions of the 3′ sequencing adaptor are deleted:
a. adaptor sequence
b. adaptor sequence permitting 1 wild-card
c. adaptor sequence permitting 1 insertion
d. adaptor sequence permitting 1 deletion
e. adaptor sequence permitting 2 deletions
f. adaptor sequence permitting 1 deletion and 1 wild-card
g. adaptor sequence permitting 1 insertion and 1 wild-card
h. adaptor sequence permitting 2 wild-cards
i. adaptor sequence permitting 3 wild-cards
j. adaptor sequence permitting 4 wild cards.
wherein: a wild-card is defined as being any 1 of the 4 deoxyribonucleic acids: (A) adenine, (T) thymine, (G) guanine, or (C) cytosine; the first nucleotide at the 5′ end of the 3′ adaptor sequence is not inserted, deleted, or subject to wild-card change, with the proviso that if the first nucleotide of the 3′ adaptor is not present, the sequence is not trimmed.
16 . The method of any one of claims 13 to 15 , 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 or the comparator cohort are candidate sRNA predictors.
17 . The method of claim 16 , 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.
18 . The method of claim 17 , wherein sequence reads are not filtered by a quality score.
19 . The method of any one of claims 13 to 18 , wherein sRNA sequences are not aligned to a reference sequence.
20 . The method of any one of claims 17 to 19 , 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.
21 . The method of claim 20 , wherein the sRNA predictors are selected that have a count of at least 50 in the majority of samples that are positive for the predictor.
22 . The method of claim 20 or 21 , wherein positive sRNA predictors are selected that are present in at least 7% of samples in the experimental cohort.
23 . The method of claim 20 or 21 , wherein positive sRNA predictors are selected that are present in at least 20% of samples in the experimental cohort.
24 . The method of claim 22 or 23 , wherein from 2 to 50 sRNA predictors are selected for inclusion in an sRNA predictor panel.
25 . The method of claim 24 , wherein from 4 to 20 sRNA predictors are selected for inclusion in an sRNA predictor panel.
26 . The method of claim 24 or 25 , wherein the presence of from 1 to 5 of the positive sRNA predictors in a sample, and optionally the absence of all of the 1 to 10 negative predictors in the sample, is indicative of the condition defined by the experimental cohort.
27 . The method of any one of claims 24 to 26 , wherein the sRNA predictors in the panel are not annotated miRNAs.
28 . The method of any one of claims 24 to 27 , further comprising, preparing a qualitative or quantitative PCR assay to detect the sRNA predictors in the panel in independent samples.
29 . A kit comprising a set of PCR primers and detectable probes for specific detection by PCR of the sRNA predictor panel identified in any one of claims 24 to 27 .
30 . The kit of claim 29 , wherein the probes comprise a fluorophore.
31 . The kit of claim 30 , wherein the probes comprise a quencher.
32 . The kit of any one of claims 29 to 31 , wherein the kit further comprises a stem-loop RT primer for amplification of the sRNA predictors.
33 . A method for determining a condition of a subject, comprising: providing a biological sample, and identifying the presence or absence of the sRNA predictor(s) identified according to the method of any one of claims 1 to 27 , or by use of the kit of any one of claims 28 to 32 , thereby determining the condition of the subject.
34 . The method of claim 33 , wherein the sample is a biological fluid sample.
35 . The method of claim 34 , wherein the biological fluid samples are selected from blood, serum, plasma, urine, saliva, or cerebrospinal fluid.
36 . The method of any one of claims 33 to 35 , wherein the condition is defined by the experimental cohort.
37 . The method of any one of claims 33 to 36 , wherein the subject is positive for the condition where the sample tests positive for one or more positive predictors, and negative for all negative predictors.
38 . The method of any one of claims 33 to 37 , wherein the patient is suspected of having or exhibits symptoms of a neurodegenerative disease, a cardiovascular disease, an inflammatory or immunological disease, or a cancer.
39 . The method of claim 38 , wherein the patient displays dementia or movement disorder.
40 . The method of claim 39 , wherein the patient is suspected of having or exhibits symptoms of a neurodegenerative disease selected from Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease, and Multiple Sclerosis.
41 . The method of any one of claims 33 to 40 , wherein the sRNA predictor(s) are identified in the biological sample by qualitative or quantitative PCR assay.
42 . The method of claim 41 , wherein the PCR assay involves a fluorescently-labeled probe.
43 . A method for classifying a mixed population of cells, comprising introducing a gene construct to the cells, the gene construct comprising an encoded protein under the regulatory control of a target site specific for a positive or negative sRNA predictor.
44 . The method of claim 43 , wherein the gene construct is introduced to the cells in vivo or ex vivo.
45 . The method of claim 43 or 44 , wherein the gene construct is a plasmid or a viral vector.
46 . The method of claim 43 or 44 , wherein the gene construct is an mRNA.
47 . The method of any one of claims 43 to 46 , wherein the target site(s) are placed in non-coding segments.
48 . The method of claim 47 , wherein the non-coding segment is a 3′ and/or 5′ UTR.
49 . The method of claim 48 , wherein the encoded protein is only expressed in biologically significant amounts when the sRNA predictor is absent from the cell.
50 . The method of any one of claims 43 to 49 , wherein the encoded protein is detectable or has a biological impact on the cell.
51 . The method of claim 50 , wherein the encoded protein is a reporter protein, a transcriptional activator, a transcriptional repressor, a pro-apoptotic protein, a pro-survival protein, a lytic protein, an enzyme, a cytokine, a growth factor, a toxin, or a cell-surface receptor.
52 . The method of any one of claims 43 to 51 , wherein the construct contains a target site specific for a negative sRNA predictor to avoid expression of the encoded protein in non-diseased cells, wherein the encoded protein optionally induces cell death or apoptosis in cells that do not express the negative predictor.
53 . The method of any one of claims 43 to 51 , wherein the construct contains a target site specific for a positive sRNA predictor to avoid expression of the encoded protein in diseased cells, wherein the encoded protein optionally protects cells from insult that do not express the positive predictor.