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 for determining abundance of nucleic acids in an initial sample comprising target nucleic acids, the method comprising:
(a) adding a starting quantity of at least 1,000 unique synthetic nucleic acids to the initial sample, wherein each of the at least 1,000 unique synthetic nucleic acids comprises (i) an identifying tag and (ii) a variable region comprising at least 5 degenerate bases;
(b) performing a sequencing assay on a portion of the target nucleic acids and on a portion of the at least 1,000 unique synthetic nucleic acids, thereby obtaining target and synthetic nucleic acid sequence reads; and
(c) calculating a diversity loss value for the portion of the at least 1,000 unique synthetic nucleic acids, wherein abundance of the target nucleic acids in the initial sample is determined using the diversity loss value.
2. The method of claim 1 , wherein the target nucleic acids comprise pathogen nucleic acids.
3. The method of claim 2 , wherein the pathogen nucleic acids are from at least five different pathogens.
4. The method of claim 1 , wherein the at least 1,000 unique synthetic nucleic acids comprise DNA.
5. The method of claim 1 , wherein each of the at least 1,000 unique synthetic nucleic acids is less than 500 base pairs or nucleotides in length.
6. The method of claim 1 , wherein the initial sample is blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchial-alveolar lavage, urine, stool, saliva, or a nasal sample.
7. The method of claim 1 , wherein the initial sample is a sample of isolated nucleic acids.
8. The method of claim 1 , further comprising generating a sequencing library from the initial sample, wherein the at least 1,000 unique synthetic nucleic acids are added to the sample before the generating of the sequencing library.
9. The method of claim 1 , wherein the diversity loss value for the portion of the at least 1,000 unique synthetic nucleic acids indicates a loss of one or more nucleic acids during sample processing of the initial sample.
10. The method of claim 1 , wherein the identifying tag of each of the at least 1,000 unique synthetic nucleic acids comprises a common sequence.
11. The method of claim 1 , wherein the initial sample is from a human subject.
12. The method of claim 1 , wherein the at least 1,000 unique synthetic nucleic acids comprise at least 10 4 unique synthetic nucleic acids.
13. The method of claim 1 , further comprising adding additional synthetic nucleic acids to the initial sample, wherein the additional synthetic nucleic acids have at least three different lengths.
14. The method of claim 1 , further comprising adding to the initial sample a first group of additional synthetic nucleic acids with a first length, a second group of additional synthetic nucleic acids with a second length, and a third group of additional synthetic nucleic acids with a third length, wherein the first group of additional synthetic nucleic acids, the second group of additional synthetic nucleic acids and third group of additional synthetic nucleic acids each comprises synthetic nucleic acids with at least three different GC contents.
15. The method of claim 13 , further comprising using the additional synthetic nucleic acids to calculate abundance of the target nucleic acids in the initial sample.
16. The method of claim 13 , further comprising using the additional synthetic nucleic acids to calculate abundance of the target nucleic acids in the initial sample based on lengths, GC contents, or both lengths and GC contents of the additional synthetic nucleic acids.
17. The method of claim 1 , wherein the at least 1,000 unique synthetic nucleic acids are added to the initial sample at a first sample processing step.
18. The method of claim 17 , further comprising adding an additional pool of at least 1,000 unique synthetic nucleic acids to the initial sample at a second sample processing step, wherein the second sample processing step is different from the first sample processing step.
19. The method of claim 18 , further comprising calculating a diversity loss value for the additional pool of at least 1,000 unique synthetic nucleic acids.
20. The method of claim 18 , further comprising identifying a sample processing step with relatively high diversity loss by comparing the diversity loss value for the at least 1,000 unique synthetic nucleic acids with the diversity loss value for the additional pool of at least 1,000 unique synthetic nucleic acids.
21. The method of claim 18 , wherein each of the unique synthetic nucleic acids in the additional pool of at least 1,000 unique synthetic nucleic acids comprises a domain identifying the synthetic nucleic acid as a member of the additional pool of at least 1,000 unique synthetic nucleic acids.
22. The method of claim 1 , further comprising adding a sample identifier nucleic acid to the initial sample.
23. The method of claim 1 , wherein (a) further comprises adding non-unique synthetic nucleic acids to the initial sample.
24. The method of claim 1 , further comprising: reporting a result of the method to a caregiver, patient, or other person.
25. The method of claim 1 , wherein the determined abundance is a relative abundance.
26. The method of claim 1 , wherein the determined abundance is absolute abundance.
27. The method of claim 1 , further comprising determining a number of synthetic nucleic acid sequence reads comprising different variable regions.
28. The method of claim 27 , wherein the diversity loss value is calculated by comparing the starting quantity of the at least 1,000 unique synthetic nucleic acids with the number of synthetic nucleic acid sequence reads comprising different variable regions.
29. The method of claim 27 , wherein the number of different synthetic nucleic acid sequence reads comprising different variable regions are detected by aligning the synthetic nucleic acid sequence reads with a reference sequence.
30. The method of claim 28 , wherein the number of different synthetic nucleic acid sequence reads comprising different variable regions is quantified by aligning the synthetic nucleic acid sequence reads with each other and throwing out duplicate reads.