Methods for detecting modification resistant nucleic acids
Methods are provided for, inter alia, detecting nucleic acid molecules resistant to degradation, such as a plurality of RNA molecules bound to a ribosome, using various technologies including deep sequencing.
1. A method for determining a protein synthesis rate, comprising:
(a) isolating a plurality of monosomes from a plurality of polysomes, wherein each of said plurality of polysomes comprises a ribosome bound to a portion of a translatable RNA molecule;
(b) sequencing each of said portions;
(c) determining a ribosome footprint density for each translatable RNA molecule from said sequencing; and
(d) determining a protein synthesis rate from the ribosomal footprint density.
2. The method of claim 1 , wherein the determining of the ribosome footprint density comprises aligning each of said portions.
3. The method of claim 1 , further comprising quantifying each of said portions.
4. The method of claim 1 , wherein said isolating comprises contacting said plurality of polysomes with a degradant.
5. The method of claim 1 , further comprising quantifying a relative amount of two or more of said portions.
6. The method of claim 2 , further comprising determining a location of high ribosome footprint density within said translatable RNA molecule.
7. The method of claim 1 , wherein said sequencing is determined by using a sequence by synthesis technique.
8. The method of claim 1 , wherein said determining of said protein synthesis rate comprises determining expression of a plurality of protein coding genes expressed from said translatable RNA molecules.
9. The method of claim 8 , wherein said protein coding genes are located on two or more different chromosomes.
10. The method of claim 4 , further comprising removing ribosomal RNA prior to step (a).
11. The method of claim 1 , further comprising amplifying the translatable RNA portion prior to sequencing.
12. A method for determining the protein synthesis rate for a plurality of protein coding genes, comprising:
(a) isolating a plurality of monosomes from a plurality of polysomes, wherein each of said plurality of polysomes comprises a plurality of ribosomes, wherein each of said plurality of ribosomes is bound to a portion of a mRNA molecule;
(b) aligning the sequence of each of said portions; and
(c) determining the ribosomal footprint density from said aligning, thereby determining the protein synthesis rate for a plurality of protein coding genes.
13. The method of claim 12 , wherein said isolating comprises comprises contacting said plurality of polysomes with a degradant.
14. The method of claim 12 , further comprising quantifying a relative amount of two or more of said portions.
15. The method of claim 12 , further comprising removing ribosomal RNA prior to step (a).
16. The method of claim 12 , further comprising determining a location of high ribosome footprint density within said mRNA molecule.
17. The method of claim 12 , wherein said sequencing is determined by using a sequence by synthesis technique.
18. The method of claim 12 , wherein said determining of said protein synthesis rate comprises determining expression of a plurality of protein coding genes expressed from said mRNA molecules.
19. The method of claim 18 , wherein said protein coding genes are located on two or more different chromosomes.
20. The method of claim 12 , further comprising amplifying the portion prior to sequencing.