Synthetic nucleic acid spike-ins
This disclosure provides methods for determining relative abundance of one or more non-host species in a sample from a host. Also provided are methods involving addition of known concentrations of synthetic nucleic acids to a sample and performing sequencing assays to identify non-host species such as pathogens. Also provided are methods of tracking samples, tracking reagents, and tracking diversity loss in sequencing assays.
1. A method of determining abundance of nucleic acids in an initial sample comprising target nucleic acids, the method comprising:
(a) adding a known quantity of spike-in nucleic acids to the initial sample comprising target nucleic acids, wherein the spike-in nucleic acids comprise at least 1,000 spike-in nucleic acids with sequences that are unique to each other;
(b) performing a sequencing assay on a portion of the target nucleic acids and a portion of the at least 1000 spike-in nucleic acids, thereby obtaining target nucleic acid sequence reads and spike-in nucleic acid sequence reads; and
(c) using the spike-in nucleic acid sequence reads to calculate abundance of the target nucleic acids.
2. The method of claim 1 , wherein the target nucleic acids comprise microbial nucleic acids.
3. The method of claim 2 , wherein the target nucleic acids comprise microbial cell-free nucleic acids from at least five different microbes.
4. The method of claim 1 , wherein the at least 1,000 spike-in nucleic acids comprise DNA.
5. The method of claim 1 , wherein at least 1,000 of the at least 1,000 spike-in nucleic acids is less than 200 base pairs or nucleotides in length.
6. The method of claim 1 , wherein at least 1,000 of the at least 1,000 spike-in nucleic acids is at least 25 base pairs in length and at most 500 base pairs in length.
7. The method of claim 1 , wherein the initial sample is blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchial-alveolar lavage, urine, stool, saliva, a nasal sample, or any combination thereof.
8. The method of claim 1 , wherein the initial sample is a plasma sample.
9. The method of claim 1 , wherein the initial sample is a sample of nucleic acids isolated from a biological sample.
10. The method of claim 1 , wherein the sequencing assay comprises generating a sequencing library, wherein the sequencing library comprises the spike-in nucleic acids and the target nucleic acids.
11. The method of claim 1 , wherein the spike-in nucleic acids comprise nucleic acids with a first length, a second length and a third length, wherein the first length, the second length, and the third length are different lengths.
12. The method of claim 1 , wherein the spike-in nucleic acids are added to DNA that has been extracted from the initial sample, wherein the initial sample is a plasma sample.
13. The method of claim 1 , wherein the initial sample is from a human.
14. The method of claim 1 , wherein the initial sample comprises DNA.
15. The method of claim 1 , wherein the sequencing assay comprises high-throughput sequencing.
16. The method of claim 15 , wherein the high-throughput sequencing comprises a sequencing-by-synthesis assay.
17. The method of claim 15 , wherein the high-throughput sequencing is next generation sequencing.
18. The method of claim 1 , wherein the using the spike-in nucleic acid sequence reads to calculate abundance of the target nucleic acids comprises using the spike-in nucleic acid reads to normalize abundance of the target nucleic acids.
19. The method of claim 1 , wherein the using the spike-in nucleic acid sequence reads to calculate abundance of the target nucleic acids comprises calculating a number of molecules of target nucleic acids per initial sample volume.
20. The method of claim 8 , wherein the target nucleic acids comprise microbial nucleic acids.
21. The method of claim 20 , wherein the target nucleic acids comprise microbial nucleic acids from at least five different microbes.
22. The method of claim 8 , wherein the at least 1,000 spike-in nucleic acids comprise DNA.
23. The method of claim 8 , wherein at least 1,000 of the at least 1,000 spike-in nucleic acids is less than 200 base pairs or nucleotides in length.
24. The method of claim 8 , wherein at least 1,000 of the at least 1,000 spike-in nucleic acids is at least 25 base pairs in length and at most 500 base pairs in length.
25. The method of claim 8 , wherein the sequencing assay comprises generating a sequencing library, wherein the sequencing library comprises the spike-in nucleic acids and the target nucleic acids.
26. The method of claim 8 , wherein the spike-in nucleic acids comprise nucleic acids with a first length, a second length and a third length, wherein the first length, the second length, and the third length are different lengths.
27. The method of claim 26 , wherein the nucleic acids with the first length, the second length, and the third length have different GC contents.
28. The method of claim 26 , further comprising using abundance of the nucleic acids with the first length, the second length and the third length to normalize abundance of the target nucleic acids.
29. The method of claim 27 , further comprising using the different GC contents to normalize abundance of the target nucleic acids.
30. The method of claim 8 , wherein the initial sample is from a human.
31. The method of claim 30 , wherein the target nucleic acids comprise microbial cell-free nucleic acids.
32. The method of claim 8 , wherein the initial sample comprises DNA.
33. The method of claim 32 , wherein the DNA comprises a mixture of human genomic DNA and microbial genomic DNA.
34. The method of claim 31 , wherein the microbial cell-free nucleic acids comprise microbial DNA.
35. The method of claim 30 , wherein the initial sample comprises bacterial DNA.
36. The method of claim 30 , wherein the initial sample comprises parasite DNA.
37. The method of claim 30 , wherein the initial sample comprises fungal DNA.
38. The method of claim 30 , wherein the initial sample comprises cell free DNA (cfDNA).
39. The method of claim 38 , wherein the sequencing assay comprises high-throughput sequencing.
40. The method of claim 39 , wherein the high-throughput sequencing comprises a sequencing-by-synthesis assay.
41. The method of claim 39 , wherein the high-throughput sequencing is next generation sequencing.
42. The method of claim 41 , wherein the method further comprises a bioinformatic analysis of the sequence reads.
43. The method of claim 41 , wherein the initial sample comprises human sequences and the bioinformatic analysis comprises sequestering or subtracting host sequences from the bioinformatic analysis of the sequence reads.
44. The method of claim 8 , wherein the using the spike-in nucleic acid sequence reads to calculate abundance of the target nucleic acids comprises using the spike-in nucleic acid reads to normalize abundance of the target nucleic acids.
45. The method of claim 8 , wherein the using the spike-in nucleic acid sequence reads to calculate abundance of the target nucleic acids comprises calculating a number of molecules of target nucleic acids per initial sample volume.
46. The method of claim 1 , wherein the initial sample is a body fluid sample.
47. The method of claim 14 , wherein the DNA comprises genomic DNA.
48. The method of claim 47 , wherein the initial sample comprises microbial DNA.
49. The method of claim 47 , wherein the genomic DNA comprises cell free DNA (cfDNA).