Nuclease-based RNA depletion
The present disclosure is related to methods and materials for depleting unwanted RNA species from a nucleic acid sample. In particular, the present disclosure describes how to remove unwanted rRNA, tRNA, mRNA or other RNA species that could interfere with the analysis, manipulation and study of target RNA molecules in a sample.
1. A method for depleting off-target RNA molecules from a nucleic acid sample comprising:
a. contacting a nucleic acid sample comprising at least one target RNA or DNA sequence and at least one off-target RNA molecule with a probe set comprising at least 40 DNA probes complementary to discontiguous sequences along the full length of the at least one off-target RNA molecule, thereby hybridizing the at least 40 DNA probes to the off-target RNA molecules to form DNA:RNA hybrids, wherein each DNA:RNA hybrid is at least 5 bases apart, or at least 10 bases apart, along a given off-target RNA molecule sequence from any other DNA:RNA hybrid, and each of the at least 40 DNA probes is 40 to 60 bases in length; and
b. contacting the DNA:RNA hybrids with a ribonuclease that degrades the RNA from the DNA:RNA hybrids, thereby degrading the off-target RNA molecules in the nucleic acid sample to form a degraded mixture, wherein the ribonuclease is hybridase.
2. The method of claim 1 , further comprising:
a. degrading any remaining DNA probes by contacting the degraded mixture with a DNase I to form a DNA degraded mixture; and
b. separating the degraded RNA from the degraded mixture or the DNA degraded mixture.
3. The method of claim 1 , wherein the contacting with the probe set comprises treating the nucleic acid sample with formamide wherein the formamide is present during the contacting with the probe set at a concentration of from about 10 to 45% by volume.
4. The method of claim 1 , wherein the contacting with the probe set comprises applying heat above the melting temperature of the at least one DNA:RNA hybrid.
5. The method of claim 1 , wherein the nucleic acid sample is from a human or from a non-human eukaryote, bacterium, virus, plant, soil, or a mixture thereof, optionally wherein the non-human eukaryote is a rat, mouse, or non-human primate.
6. The method of claim 1 , wherein the off-target RNA is rRNA and/or globin mRNA.
7. The method of claim 1 , wherein the probe set comprises at least two DNA probes that hybridize to at least one off-target RNA molecule selected from:
a. 28S, 18S, 5.8S, 5S, 16S, and/or 12S from humans;
b. HBA-A1, HBA-A2, HBB, HBG1, and HBG2 from hemoglobin, and 23S, 16S, and 5S from Gram positive or Gram negative bacteria; and/or
c. rat 16S, rat 28S, mouse 16S, and mouse 28S, and combinations thereof.
8. The method of claim 1 , wherein the probe set comprises DNA probes to a particular off-target RNA molecule that are complementary to about 80 to 85% of the sequence of the off-target RNA molecule,
optionally wherein the DNA probes comprise:
a. two or more sequences selected from SEQ ID NOs: 1-333; or
b. two or more sequences selected from SEQ ID NOs: 1-428; or
c. two or more sequences selected from SEQ ID NOs: 1-377; or
d. two or more sequences selected from SEQ ID NOs: 1-333 and SEQ ID NOs: 378-428; or
e. two or more sequences selected from SEQ ID NOs: 334-377; or
f. two or more sequences selected from SEQ ID NOs: 378-428;
or a combination thereof.
9. A composition comprising
a. a probe set comprising at least 40 DNA probes complementary to discontiguous sequences along the full length of at least one off-target RNA molecule in a nucleic acid sample, each DNA probe is hybridized at least 5, or at least 10, bases apart along the full length of the at least one off-target RNA molecule from any other DNA probe in the probe set, and each DNA probe is 40 to 60 bases in length; and
b. a ribonuclease capable of degrading RNA in a DNA:RNA hybrid, wherein the ribonuclease is a hybridase.
10. The composition of claim 9 , further comprising DNase I for degrading any remaining DNA probes.
11. The composition of claim 9 , further comprising formamide wherein the formamide is present at about 10 to 45% by volume when the DNA probes come in contact with the nucleic acid sample.
12. The composition of claim 9 , wherein the off-target RNA is rRNA and/or globin mRNA.
13. The composition of claim 9 , wherein the probe set comprises at least two DNA probes that hybridize to at least one off-target RNA molecule selected from:
a. 28S, 18S, 5.8S, 5S, 16S, and/or 12S from humans;
b. HBA-A1, HBA-A2, HBB, HBG1, and HBG2 from hemoglobin, and 23S, 16S, and 5S from Gram positive or Gram negative bacteria; and/or
c. rat 16S, rat 28S, mouse 16S, and mouse 28S, and combinations thereof.
14. The composition of claim 9 , wherein the probe set comprises DNA probes comprising:
a. two or more sequences selected from SEQ ID NOs: 1-333; or
b. two or more sequences selected from SEQ ID NOs: 1-428; or
c. two or more sequences selected from SEQ ID NOs: 1-377; or
d. two or more sequences selected from SEQ ID NOs: 1-333 and SEQ ID NOs: 378-428; or
e. two or more sequences selected from SEQ ID NOs: 334-377; or
f. two or more sequences selected from SEQ ID NOs: 378-428;
g. or a combination thereof.
15. A kit comprising:
a. a probe set comprising at least 40 DNA probes complementary to discontiguous sequences at least 5, or at least 10, bases apart along the full length of at least one off-target RNA molecule in a nucleic acid sample, and each DNA probe is 40 to 60 bases in length;
b. a ribonuclease capable of degrading RNA in a DNA:RNA hybrid, wherein the ribonuclease is hybridase; and
c. DNase I capable of degrading any remaining DNA probes; and
d. formamide wherein the formamide is present during the contacting with the probe set at a concentration of from about 10 to 45% by volume.
16. The kit of claim 15 , wherein the off-target RNA is rRNA and/or globin mRNA.
17. The kit of claim 15 , wherein the probe set comprises at least two DNA probes that hybridize to at least one off-target RNA molecule selected from:
a. 28S, 18S, 5.8S, 5S, 16S, and/or 12S from humans;
b. HBA-A1, HBA-A2, HBB, HBG1, and HBG2 from hemoglobin, and 23S, 16S, and 5S from Gram positive or Gram negative bacteria; and/or
c. rat 16S, rat 28S, mouse 16S, and mouse 28S, and combinations thereof.
18. The kit of claim 15 , wherein the probe set comprises DNA probes to a particular off-target RNA molecule that are complementary to about 80 to 85% of the sequence of the off-target RNA molecule,
optionally wherein the DNA probes comprise:
a. two or more-sequences selected from SEQ ID NOs: 1-333; or
b. two or more sequences selected from SEQ ID NOs: 1-428; or
c. two or more sequences selected from SEQ ID NOs: 1-377; or
d. two or more sequences selected from SEQ ID NOs: 1-333 and SEQ ID NOs: 378-428; or
e. two or more sequences selected from SEQ ID NOs: 334-377; or
f. two or more sequences selected from SEQ ID NOs: 378-428;
or a combination thereof.
19. The kit of claim 15 , further comprising:
a. a probe set comprising SEQ ID NOs: 1-333; and
b. RNA purification beads.
20. A method of supplementing a probe set for use in depleting off-target RNA nucleic acid molecules from a nucleic acid sample comprising:
a. contacting a nucleic acid sample comprising at least one RNA or DNA target sequence and at least one off-target RNA molecule from a first species with a probe set comprising at least 40 DNA probes complementary to discontiguous sequences along the full length of the at least one off-target RNA molecule from a second species, thereby hybridizing the at least two DNA probes to the off-target RNA molecules to form DNA:RNA hybrids, wherein
i. each DNA:RNA hybrid is at least 5 bases apart, or at least 10 base apart, along a given off-target RNA molecule sequence from any other DNA:RNA hybrid; and
ii. each DNA probe is 40 to 60 bases in length;
b. contacting the DNA:RNA hybrids with hybridase that degrades the RNA from the DNA:RNA hybrids, thereby degrading the off-target RNA molecules in the nucleic acid sample to form a degraded mixture;
c. separating the degraded RNA from the degraded mixture;
d. sequencing the remaining RNA from the sample;
e. evaluating the remaining RNA sequences for the presence of off-target RNA molecules from the first species, thereby determining gap sequence regions; and
f. supplementing the probe set with additional DNA probes complementary to discontiguous sequences in one or more of the gap sequence regions.
21. The method of claim 20 , wherein the composition of claim 9 is used to supply the hybridase and the probe set comprising the DNA probes complementary to discontiguous sequences along the full length of the at least one off-target RNA molecule of a human.
22. The method of claim 20 , wherein the method is used to identify DNA probes that hybridize to one or more off-target RNA molecules from rat and/or mouse, optionally selected from rat 16S, rat 28S, mouse 16S, and mouse 28S, and combinations thereof.