Methods and systems for sequencing polynucleotides
View Patent ↗The present disclosure relates generally to detection on non-coding RNAS molecules in a sample or diagnosis of subject based upon detection or quantification of non-coding nucleic acid sequences in a sample, specifically to identify and use of molecular biomarkers for cancer including breast cancer.
1. A method for sequencing a non-coding ribonucleic acid (RNA) molecule from a cell-free sample obtained from a human female subject, comprising:
(a) providing the cell-free sample from the human female subject, wherein the cell-free sample comprises a first set of non-coding RNA molecules;
(b) subjecting the first set of non-coding RNA molecules to reverse transcription to generate one or more complementary deoxyribonucleic acid (cDNA) molecules;
(c) sequencing the one or more cDNA molecules or derivatives thereof; and
(d) based on the sequencing in (c), determining whether the first set of non-coding RNA molecules contains one or more non-coding RNA molecules of a second set of non-coding RNA molecules, wherein the second set of non-coding RNA molecules are present in breast tumor samples, and wherein the second set of non-coding RNA molecules have a 90 th percentile expression in normal samples below 0.5 cpm (count-per-million) reads.
2. The method of claim 1 , wherein the non-coding RNA molecule has a length of less than 200 nucleotides.
3. The method of claim 1 , wherein the non-coding RNA molecule has a length between 50 and 100 nucleotides.
4. The method of claim 1 , further comprising, after (b), amplifying a cDNA molecule of the one or more cDNA molecules.
5. The method of claim 1 , further comprising, after (a), isolating the first set of non-coding RNA molecules from other components of the cell-free sample.
6. The method of claim 5 , wherein the isolating comprises filtration.
7. The method of claim 1 , further comprising using a result of the sequencing to determine an amount of a non-coding sequence of a non-coding RNA molecule of the first set of non-coding RNA molecules in the cell-free sample.
8. The method of claim 1 , wherein the cell-free sample comprises serum.
9. The method of claim 1 , wherein the cell-free sample comprises plasma.
10. The method of claim 1 , wherein the cell-free sample comprises urine.
11. The method of claim 1 , wherein the cell-free sample comprises lymph.
12. The method of claim 1 , wherein the cell-free sample comprises saliva.
13. The method of claim 1 , wherein a total volume of the cell-free sample is between 20 microliters and 2 milliliters.
14. The method of claim 1 , wherein a total volume of the cell-free sample is between 100 microliters to 500 microliters.
15. The method of claim 1 , wherein the cell-free sample comprises plasma, and wherein a total volume of the cell-free sample is between 100 microliters and 1 milliliter.
16. The method of claim 1 , wherein the sequencing in (c) generates sequencing reads, and wherein the sequencing reads are processed to identify a non-coding sequence of a non-coding RNA molecule of the first set of non-coding RNA molecules.
17. The method of claim 1 , wherein the sequencing in (c) comprises sequencing-by-synthesis.
18. The method of claim 1 , wherein the normal samples are from subjects who do not have breast cancer.
19. The method of claim 4 , wherein the amplifying comprises performing a polymerase chain reaction (PCR).
20. The method of claim 4 , wherein the amplifying comprises rolling circle amplification.