Profiling expression at transcriptome scale
Methods for designing oligonucleotide probes for detecting and profiling expression products at transcriptome scale.
1. A method for selectively detecting target nucleic acid sequences of a plurality of full-length sequences of interest (FLSs) in a sample, wherein the FLSs are members of a defined set of full-length species that may be present in the sample, comprising the steps of
(a) selecting a target sequence for each FLS by
(1) defining a plurality of subsequences of the FLS as target sequence candidates (TSCs),
wherein each TSC has a downstream region (DR) and an upstream region (UR),
wherein the DR has ligation-selective bases (DLSB) at the 5′-end, or the UR has ligation-selective bases (ULSB) at the 3′-end, or both, collectively being the ligation-selective bases (TSC-LSB),
wherein the sequence of the TSC-LSB spans an exon or splice junction;
(2) identifying a similar full-length species (SFL) in the defined set that comprises regions that are similar to regions in the TSC, wherein the SFL has ligation-selective bases (SFL-LSB) that correspond to the ligation-selective bases of the target sequence candidate (TSC-LSB);
(3) selecting a TSC to be the target sequence based on the following factors:
(i) the difference of one or more bases of the TSC-LSB to the corresponding bases of the SFL-LSB;
(b) contacting the sample with pairs of target sequence oligos, each pair comprising
(1) a downstream detector oligo (DD) comprising a portion (DR′) complementary to the DR of the selected TSC, and
(2) an upstream detector oligo (UD) comprising a portion (UR′) complementary to the UR of the selected TSC;
(c) ligating the DD and UD if both are hybridized to the DR and UR of a target sequence in the sample;
whereby the ligation product indicates detection of the target sequence that is selective over SFLs in the sample.
2. The method of claim 1 , wherein the defined set of full-length species is the human transcriptome.
3. The method of claim 2 , wherein the defined set has at least 500 species.
4. The method of claim 1 , wherein the number of target nucleic acid sequences is at least 500.
5. The method of claim 1 , wherein the TSC is at least 20 bases in length.
6. The method of claim 1 , wherein the TSC-LSB is at least 2 or more bases in length.
7. The method of claim 1 , wherein a TSC is preferentially selected that is dissimilar from all other sequences in the defined set.
8. The method of claim 7 , wherein the dissimilarity is determined by a blastn comparison.
9. The method of claim 1 , wherein the degree of similarity in step (a)(3)(i) is based on a predetermined set of values for patterns of complementary bases.
10. The method of claim 1 , wherein a factor of step (a)(3) is
(ii) penalizing a TSC if the 25th base in the 5′-to-3′ direction is a C.
11. The method of claim 1 , wherein a factor of step (a)(3) is
(ii) penalizing a TSC for distance from the 3′-end of the FLS.
12. The method of claim 1 , wherein a cluster of isoforms is identified from members of the defined set that share a common sequence, and the defined set excludes the isoforms of the cluster.
13. The method of claim 12 , wherein the FLS is the longest isoform of the cluster.
14. The method of claim 12 , wherein a factor of step (a)(3) is
(ii) penalizing a TSC for being present in fewer than all the isoforms of the cluster.
15. The method of claim 1 , wherein a factor of step (a)(3) is
(ii) penalizing a TSC if a detector oligo has a homopolymeric run of 2 or more bases.
16. The method of claim 1 , wherein a factor of step (a)(3) is
(ii) penalizing a TSC if a detector oligo has more than 2 repeated dinucleotides.
17. The method of claim 1 , wherein a factor of step (a)(3) is
(ii) penalizing a TSC if the GC % of a detector oligo is less than 40% or greater than 60%.
18. The method of claim 1 , wherein the DR and UR are separated by at least one nucleotide, and the method further comprises (b1) extending the DR′ using the sample as template.
19. The method of claim 1 , wherein the sample is provided in liquid phase.
20. The method of claim 1 , wherein the sample is provided attached to a solid substrate.
21. The method of claim 20 , wherein the sample is formalin-fixed paraffin-embedded (FFPE) tissue or cells.
22. The method of claim 20 , wherein steps (b) and (c) are performed on the sample in situ.
23. The method of claim 1 , wherein in step (b)(2), the lengths of the DR′ and UR′ are unequal but are tuned for similar % GC, annealing, or melting temperatures.