Methods for identifying and using small rna predictors

Abstract

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.