Integrated capture and amplification of target nucleic acid for sequencing
View Patent ↗The invention provides efficient methods of preparing a target nucleic acid in a form suitable for sequencing. The methods are particularly amenable for preparing high quality nucleic acids for massively parallel sequencing. The methods involve capturing a target nucleic acid from a sample and PCR amplification of the target nucleic acid. The target nucleic acid is captured by binding to a capture probe, which in turn binds to an immobilized probe. The immobilized probe is typically immobilized via a magnetic bead. The captured target nucleic acid is PCR amplified by thermocycling without prior dissociation of the target nucleic acid from the beads. The efficiency of the method lies in part in that both the capture and amplification steps are performed in a single vessel. The amplified nucleic acid can then be sequenced.
1. A method of preparing a target nucleic acid, comprising
contacting a target nucleic acid with a capture probe and an immobilized probe, the capture probe comprising a first segment that hybridizes to the target nucleic acid and a second segment that hybridizes to the immobilized probe, wherein the target nucleic acid hybridizes to the first segment of the capture probe, and the second segment of the capture probe hybridizes to the immobilized probe, thereby forming a capture hybrid capturing the target nucleic acid; and
performing a PCR amplification of the captured target nucleic acid without a step of dissociating the capture hybrid before initiating thermocycling in the PCR amplification; wherein the PCR amplification is performed in the same vessel as the contacting step and wherein the amplified target nucleic acid comprises at least one mutation that is present in less than 10% of molecules of the target nucleic acid.
2. The method of claim 1 , further comprising sequencing the amplified target nucleic acid.
3. The method of claim 1 , wherein the target nucleic acid is an RNA molecule and the PCR amplification is an RT-PCR amplification.
4. The method of claim 3 , wherein the target nucleic acid is a population of RNA molecules, and the RT-PCR amplification results in an amplified population of nucleic acids, which are sequenced in the sequencing step.
5. The method of claim 4 , wherein the target nucleic acid is a viral RNA population.
6. The method of claim 5 , wherein the viral mRNA population includes viral mRNA and/or viral genomic RNA wherein species of the viral RNA population differ from one another by mutations, which are identified by the sequencing step or wherein the identified mutations include at least one drug resistance mutation.
7. The method of claim 1 , wherein at least one identified mutation is present in less than 1% of molecules in the population of mRNA molecules.
8. The method of claim 7 , wherein the viral RNA population is an HIV, HCV or HBV mRNA population from a patient sample.
9. The method of claim 1 , wherein the immobilized probe is immobilized via attachment to a magnetic bead.
10. The method of claim 9 , wherein the concentration of immobilized probe linked to magnetic beads is 10-30 pg/ml.
11. The method of claim 1 , wherein the PCR involves thermocycling between temperature ranges of 90-99° C. and 55-65° C., or 95° C. and 55-65° C.; or wherein the concentration of the capture probe is 0.2-0.8 pmol/ml.
12. The method of claim 1 , wherein the sequencing step sequences at least 75% of the length of the target nucleic acid or wherein the RT-PCR is performed with a pair of primers hybridizing to conserved regions of the target molecule or its complement and proximate to the ends of the target molecule so as to allow amplification of at least 75% of the target molecule.
13. The method of claim 2 , wherein at least 100,000 molecules in the population of the target molecules are sequenced.
14. The method of claim 1 , wherein the target nucleic acid is present in a serum or plasma sample.
15. The method of claim 14 , wherein the serum or plasma sample is treated with detergent to release viral RNA.
16. The method of claim 1 , wherein the first segment includes a nucleic acid of 10-30 bases complementary to the target nucleic acid.
17. The method of claim 1 , wherein the first segment is complementary to a conserved region of a viral RNA target.
18. The method of claim 5 , wherein the contacting is performed with a plurality of capture probes, the capture probes having the same second segment and different first segments, the different first segments being complementary to different conserved regions of a viral RNA target.
19. The method of claim 1 , wherein the first segment includes a random sequence of nucleotides that hybridizes nonspecifically to the target nucleic acid.
20. The method of claim 1 , wherein the second segment includes a nucleic acid of 10-30 bases complementary to a nucleic acid of 10-30 contiguous bases in the immobilized probe.
21. The method of claim 20 , wherein the nucleic acid of the second segment is a homopolymer and the nucleic acid of the immobilized probe constitute a complementary homopolymer.
22. The method of claim 21 , wherein the homopolymer of the second segment is poly-A and the homopolymer of the immobilized probe is poly-T or vice versa.
23. The method of claim 1 , wherein the second segment of the capture probe and the complementary segment of the immobilized probe are L-nucleic acids.
24. The method of claim 1 , wherein the target nucleic acid is contacted with the capture probe and immobilized probe simultaneously.
25. The method of claim 1 , wherein the target nucleic acid is contacted with the capture probe before the immobilized probe.
26. The method of claim 1 , wherein the binding of the target nucleic acid to the capture probe occurs under first hybridization conditions and the binding of the capture probe to the immobilized probe occurs under second hybridization conditions and the first conditions are more stringent than the second conditions.
27. The method of claim 26 , wherein the first conditions are at a higher temperature than the second conditions.
28. The method of claim 26 , wherein the first conditions include a temperature of 50-70° C. and the second conditions include room temperature.
29. The method of claim 2 , wherein the sequencing is performed by single-molecule real-time sequencing.
30. The method of claim 1 , further comprising forming a circular template comprising the amplified target nucleic acid wherein the sequencing generates a sequencing read containing multiple copies of the target nucleic acid.