Attenuators

1. A method for detecting a plurality of target nucleic acid sequences of interest in a sample, wherein each target sequence has an upstream region (UR) and a downstream region (DR), the method comprising:

(a) contacting the sample with a pool of detector oligos comprising, for each target sequence,

(1) a downstream detector oligo (DD) comprising a portion complementary to the downstream region of the target sequence (DR′), and

(2) an upstream detector oligo (UD) comprising a portion complementary to the upstream region of the target sequence (UR′),

wherein, for a predetermined set of potentially high abundance target sequences (HATs) in the sample, also contacting the sample with

(3) an attenuator oligonucleotide that comprises a portion at least partially complementary to one of the downstream or upstream regions of a HAT, and further comprises a nonextendable or nonligatable blocking group, or a nonamplifiable segment; and

(b) ligating a downstream detector oligo for a target sequence to a corresponding upstream detector oligo if both are hybridized to the downstream and upstream regions of a target sequence in the sample;

thereby attenuating the number of HAT products to be detected.

2. The method of claim 1 , wherein the sample is provided on a solid substrate.

3. The method of claim 1 , wherein the sample is formalin-fixed paraffin-embedded (FFPE) tissue or cells.

4. The method of claim 1 , wherein at least one of the detector oligos is detectably labeled.

5. The method of claim 1 , wherein at least one of the detector oligos has a barcode sequence.

6. The method of claim 1 , wherein steps (a) and (b) are performed on the sample in situ.

7. The method of claim 1 , wherein after step (b), the ligated oligos are eluted from the sample and collected.

8. The method of claim 1 , further comprising the step of

(c) amplifying the ligated oligos.

9. The method of claim 8 , wherein the attenuator further comprises an amplification sequence.

10. The method of claim 9 , wherein the amplified products incorporate a barcode sequence that is specific to the sample.

11. The method of claim 8 , further comprising:

(d) detecting the amplified products by sequencing.

12. The method of claim 8 , further comprising:

(d) detecting the amplified products, whereby a lower abundance target sequence is detected at a higher level.

13. The method of claim 1 , further comprising

(d) detecting the ligated products by sequencing.

14. The method of claim 1 , wherein the set of HATs contains a sequence partially encoding a product selected from the group consisting of myoglobins, actins, tubulins, ubiquitins, heat-shock proteins (HSPs), ribosomal proteins, ribosomal RNAs (rRNAs), micro-RNAs (miRNAs), and small nuclear RNAs (snRNAs).

15. The method of claim 1 , wherein the set of HATs contains a sequence partially encoding a product selected from the group consisting of cytochrome c, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L7 (RPL7), ribosomal protein S6 (rpS6), snRNA RNUs, phosphoglycerokinase (PGK), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), beta-actin, and beta-tubulin.

16. The method of claim 1 , wherein the set of HATs contains a sequence partially encoding a product selected from the group consisting of alpha-2-microglobulin, vimentin, and fibronectins.

17. The method of claim 1 , wherein a HAT is a sequence partially encoding a cytochrome selected from the group consisting of mitochondrially encoded cytochrome b (MT-CYB), outer mitochondrial membrane cytochrome b5 type B, microsomal cytochrome b5 type A (ACYB5A), and ascorbate-dependent cytochrome b3 (CYBASC3).

18. The method of claim 1 , wherein an attenuator further has a portion that is complementary to a region adjacent to the downstream or upstream region of a HAT.

19. The method of claim 18 , wherein the downstream and upstream regions of the HAT are separated by one or more nucleotides, and the attenuator has a portion that is complementary to the one or more nucleotides.

20. The method of claim 1 , wherein an attenuator has a portion that is complementary to at least a portion of the downstream region of a HAT and another portion that is complementary to at least a portion of the upstream region of the HAT.

21. The method of claim 20 , wherein the attenuator hybridizes by circularization.

22. The method of claim 1 , wherein the attenuator has at least one mismatched base relative to the DR or UR of the HAT.

23. The method of claim 22 , wherein the attenuator has a portion partially complementary to a downstream region of a HAT, and has 1, 2, or 3 mismatched bases at any of the positions at the 3′ end or at 1 or 2 positions from the 3′ end.

24. The method of claim 22 , wherein the attenuator has a portion partially complementary to an upstream region of a HAT, and has 1, 2, or 3 mismatched bases at any of the positions at the 5′ end or at 1 or 2 positions from the 5′ end.

25. The method of claim 1 , wherein the blocking group is selected from the group comprising 2′,3′-dideoxynucleotide, 3′-propyl spacer, 3′-dehydroxylation, and 3′-amination.

26. The method of claim 1 , wherein the blocking group is a nonhybridizing portion.

27. The method of claim 26 , wherein the nonhybridizing portion of an attenuator forms a hairpin loop.

28. The method of claim 1 , wherein the attenuator has a selectively cleavable site.

29. The method of claim 1 , wherein the attenuator oligonucleotide comprises a portion at least partially complementary to one of the downstream or upstream regions of a HAT, and further comprising a portion at least partially complementary to the other region of the HAT.

30. The method of claim 1 , wherein the attenuator oligonucleotide comprises a nonextendable blocking group.

31. The method of claim 1 , wherein the attenuator oligonucleotide comprises a nonligatable blocking group.

32. The method of claim 1 , wherein the attenuator oligonucleotide comprises a nonamplifiable segment.

33. The method of claim 32 , wherein the nonamplifiable segment comprises a reverse complementary sequence.

34. The method of claim 32 , wherein the nonamplifiable segment comprises a nonamplifiable tail.

35. The method of claim 32 , wherein the nonamplifiable segment is RNA.

36. The method of claim 1 , wherein the sample is provided in liquid phase.

37. The method of claim 1 , further comprising

(d) detecting the ligated products, whereby detection of a lower abundance target sequence is detected at a higher level.