Systems and methods for sequencing of cell-free RNA

Abstract

Systems and methods for sequencing of cell-free RNAs are provided. Targeted sequencing can be performed using a panel of transcripts that are genes of rare abundance within a population of control samples of cell-free nucleic acids.

Description

Abstract This invention provides systems and methods for sequencing cell-free RNA. It enables targeted sequencing of gene transcriptomes that are rare in a population of cell-free nucleic acid control samples.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing for sequencing of cell-free RNA, comprising: providing a sample comprising nucleic acids for sequencing, wherein the nucleic acids for sequencing are cell-free RNA or nucleic acids derived from and representative of cell-free RNA; and contacting the sample with a panel of nucleic acid molecules that comprises molecules having sequences from or complement to gene transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies such that the panel of nucleic acid molecules anneals with a subset of the nucleic acids for sequencing.

2. The method of claim 1, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are expressed in less than 50% of a population of control liquid biopsies.

3. The method of claim 2, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are expressed in less than 5% of a population of control liquid biopsies.

4. The method of any one of claims 1-3, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are in the bottom 60% of genes with respect to normalized expression across a population of control liquid biopsies.

5. The method of claim 4, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are in the bottom 30% of genes with respect to normalized expression across the population of control liquid biopsies.

6. The method of any one of claims 1-5, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts having log transformed and normalized expression values less than zero across a population of control liquid biopsies.

7. The method of any one of claims 2-6, wherein the population of control liquid biopsies comprises at least 5 liquid biopsies.

8. The method of claim 7, wherein the population of control liquid biopsies comprises at least 50 liquid biopsies.

9. The method of any one of claims 1-8, wherein the control liquid biopsies are collected from individuals not having one or more the following when the biopsy is collected: an observed pathogenic infection, a diagnosed cancer, a diagnosed metabolic disorder, a diagnosed neurological disorder, a diagnosed immunodeficiency disorder, a diagnosed autoimmune disorder, a diagnosed inflammatory disorder, a diagnosed cardiovascular disorder, a diagnosed renal disorder, a diagnosed hepatic disorder, active pregnancy, a diagnosed pregnancy complication, a diagnosed fetal complication, an organ transplant, active rejection of an organ transplant, obesity, malnourishment, cachexia, and an abnormality on a clinical test.

10. The method of any one of claims 1-9, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies comprise 50% of transcripts from Table 3.

11. The method of claim 10, wherein the transcripts that are rarely abundant as cell- free RNA molecules within control liquid biopsies comprise 90% of transcripts from Table 3.

12. The method of claim 11, wherein the transcripts that are rarely abundant as cell- free RNA molecules within control liquid biopsies comprise 100% of transcripts from Table 3.

13. The method of any one of claims 1-12, wherein the panel of nucleic acid molecules excludes at least 50% of whole-exome gene transcripts that are not transcripts that are rarely abundant as cell-free RNA molecules.

14. The method of claim 13, wherein the panel of nucleic acid molecules excludes at least 90% of whole-exome gene transcripts that are not transcripts that are rarely abundant as cell-free RNA molecules.

15. The method of any one of claims 1-14, wherein the panel of nucleic acid molecules consists of 5000 or fewer gene transcripts in addition to transcripts that are rarely abundant as cell-free RNA molecules.

16. The method of claim 15, wherein the panel of nucleic acid molecules consists of 500 or fewer gene transcripts in addition to transcripts that are rarely abundant as cell- free RNA molecules.

17. The method of any one of claims 1-16, wherein the panel of nucleic acid molecules further comprises one or more of: tissue-specific transcripts, cell-type-specific transcripts, clinically relevant transcripts, B-cell receptor and T-cell receptor transcripts, biomarkers, commonly mutagenized transcripts and a set of control transcripts for normalization between samples.

18. The method of claim 17, wherein the biomarkers are associated with one of the following biological characteristics: a medical disorder, pregnancy, a fetal complication, a pregnancy complication, a neoplastic growth, cancer, a particular cancer type, a pathogen infection, immune activation, organ transplant rejection, neurodegeneration, tissue of origin, cell type of origin, or activation of a biochemical pathway.

19. The method of any one of claims 1-18, wherein the panel of nucleic acid molecules are a set of probes for targeted capture hybridization.

20. The method of any one of claims 1-18, wherein the panel of nucleic acid molecules are a set of primers for targeted amplification.

21. The method of any one of claims 1-19, wherein the sample of cfRNA is derived from blood, plasma, lymph, cerebrospinal fluid, amniotic fluid, urine, or stool.

22. The method of claim 1 further comprising: generating a sequencing library derived from the sample; and performing targeted sequencing of the sequencing library to yield a sequencing result of the cell-free RNA, wherein the sequencing is targeted towards the panel of nucleic acid molecules.

23. The method of claim 22, further comprising: removing platelet expression from the sequencing result in silico.

24. The method of claim 22 or 23 further comprising: performing differential transcript analysis with the sequencing result and a second sequencing result.

25. The method of any one of claims 22-24 further comprising: detecting enrichment of at least one expression signature within the sequencing result.

26. The method of any one of claims 22-25 further comprising: detecting sequence mutagenesis within the sequencing result.

27. The method of any one of claims 22-26 further comprising: inferring copy number status of one or more genes from the sequencing result.

28. The method of any one of claims 22-27 further comprising: utilizing the sequencing result along with a plurality of other sequencing results to train a computational model to predict a categorical status or a likelihood of a biological characteristic, wherein the cell-free RNA sample has a known categorical status of a biological characteristic.

29. The method of any one of claims 22-27 further comprising: utilizing the sequencing result as input within a trained computational model to predict a categorical status or a likelihood of a biological characteristic, wherein the computational model has been trained utilizing a cohort of RNA sequencing results having a known categorical status of a biological characteristic.

30. The method of any one of claims 22-27 further comprising: deriving one or more features from the sequencing result, wherein the one or features comprises enrichment of one or more gene signatures, enrichment of biochemical pathways, collection of sequence variants, and copy number status; and utilizing the one or more derived features as input within a trained computational model to predict a categorical status or a likelihood of a biological characteristic, wherein the computational model has been trained utilizing a cohort of RNA sequencing results having a known categorical status of a biological characteristic.

31. A panel of nucleic acids for targeting transcripts that are rarely abundant as cell- free RNA molecules, the panel comprising: nucleic acid molecules having sequences from or complement to gene transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies.

32. The panel of nucleic acids of claim 31, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are expressed in less than 50% of a population of control liquid biopsies.

33. The panel of nucleic acids of claim 32, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are expressed in less than 5% of a population of control liquid biopsies.

34. The panel of nucleic acids of any one of claims 31-33, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are in the bottom 60% of genes with respect to normalized expression across a population of control liquid biopsies.

35. The panel of nucleic acids of claim 34, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts that are in the bottom 30% of genes with respect to normalized expression across the population of control liquid biopsies.

36. The panel of nucleic acids of any one of claims 31-35, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies are defined as transcripts having log transformed and normalized expression values less than zero across a population of control liquid biopsies.

37. The panel of nucleic acids of any one of claims 32-36, wherein the population of control liquid biopsies comprises at least 5 liquid biopsies.

38. The panel of nucleic acids of claim 37, wherein the population of control liquid biopsies comprises at least 50 liquid biopsies.

39. The panel of nucleic acids of any one of claims 31-38, wherein the control liquid biopsies are collected from individuals not having one or more the following when the biopsy is collected: an observed pathogenic infection, a diagnosed cancer, a diagnosed metabolic disorder, a diagnosed neurological disorder, a diagnosed immunodeficiency disorder, a diagnosed autoimmune disorder, a diagnosed inflammatory disorder, a diagnosed cardiovascular disorder, a diagnosed renal disorder, a diagnosed hepatic disorder, active pregnancy, a diagnosed pregnancy complication, a diagnosed fetal complication, an organ transplant, active rejection of an organ transplant, obesity, malnourishment, cachexia, and an abnormality on a clinical test.

40. The panel of nucleic acids of any one of claims 31-39, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies comprise 50% of transcripts from Table 3.

41. The panel of nucleic acids of claim 40, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies comprise 90% of transcripts from Table 3.

42. The panel of nucleic acids of claim 41, wherein the transcripts that are rarely abundant as cell-free RNA molecules within control liquid biopsies comprise 100% of transcripts from Table 3.

43. The panel of nucleic acids of any one of claims 31-42, wherein the panel of nucleic acid molecules excludes at least 50% of whole-exome gene transcripts that are not transcripts that are rarely abundant as cell-free RNA molecules.

44. The panel of nucleic acids of claim 43, wherein the panel of nucleic acid molecules excludes at least 90% of whole-exome gene transcripts that are not transcripts that are rarely abundant as cell-free RNA molecules.

45. The panel of nucleic acids of any one of claims 31-44, wherein the panel of nucleic acid molecules consists of 5000 or fewer gene transcripts in addition to transcripts that are rarely abundant as cell-free RNA molecules.

46. The panel of nucleic acids of claim 45, wherein the panel of nucleic acid molecules consists of 500 or fewer gene transcripts in addition to transcripts that are rarely abundant as cell-free RNA molecules.

47. The panel of nucleic acids of any one of claims 31-46, wherein the panel of nucleic acid molecules further comprises tissue-specific transcripts, cell-type-specific transcripts, clinically relevant transcripts, B-cell receptor and T-cell receptor transcripts, biomarkers, and commonly mutagenized transcripts.

48. The panel of nucleic acids of claim 47, wherein the biomarkers are associated with one of the following biological characteristics: a medical disorder, pregnancy, a fetal complication, a pregnancy complication, a neoplastic growth, cancer, a particular cancer type, a pathogen infection, immune activation, organ transplant rejection, neurodegeneration, tissue of origin, cell type of origin, or activation of a biochemical pathway.

49. The panel of nucleic acids of any one of claims 31-48, wherein the panel of nucleic acid molecules further comprises a set of control transcripts for normalization between samples.

50. The panel of nucleic acids of any one of claims 31-49, wherein the panel of nucleic acid molecules are a set of probes for targeted capture hybridization.

51. The panel of nucleic acids of any one of claims 31-49, wherein the panel of nucleic acid molecules are a set of primers for targeted amplification.

52. A method of extracting RNA from a cell-free source, comprising: (a) adding glycogen to a sample comprising cell-free nucleic acids; and (b) contacting a silica-based column with a sample comprising cell-free nucleic acids.

53. The method of claim 52, wherein step (a) is performed before step (b).

54. The method of claim 52 or 53 further comprising: eluting cell-free nucleic acids from the silica-based column to yield a solution of extracted cell-free nucleic acids; and contacting the solution of extracted cell-free nucleic acids with a DNase.

55. A method of quantifying cell-free RNA for downstream molecular applications, comprising: providing a sample comprising cell-free RNA; reverse transcribing the cell-free RNA to yield cDNA; and quantifying the concentration of cell-free RNA within the solution using quantitative real-time polymerase chain reaction and the cDNA.

56. The method of claim 55, wherein the step of quantifying the concentration of cell- free RNA further comprises generating a standard curve based on a set of control standards having known concentration, wherein the control standards are also assessed using quantitative real-time polymerase chain reaction.

57. The method of claim 55 or 56, wherein the sample further comprises cell-free DNA, the method further comprising: quantifying the concentration of cell-free DNA within the sample using quantitative real-time polymerase chain reaction, wherein the cell-free RNA is quantified by using primers that span across an intron of a gene that is relatively stable across cell-free RNA samples and the cell-free DNA is quantified by using primers that anneal to atranscriptionally silent region of a genome that is relatively stable across cell-free DNA samples.

58. The method of claim 57, wherein the primers for quantifying cell-free RNA span across an intron of GAPDH and the primers for quantifying cell-free DNA target cover a 78bp transcriptionally silent region of chromosome 12.

59. A method of sequencing cell-free RNA, comprising: providing a library of nucleic acid molecules, wherein the library of nucleic acid molecules was derived from cell-free RNA, wherein the cell-free RNA is derived from a liquid biopsy; sequencing the library of nucleic acid molecules to yield a sequencing result; and removing variation due to transcript expression associated with platelets.

60. The method of claim 59, wherein the library of nucleic acid molecules was generated by capturing or amplifying nucleic acid molecules.

61. The method of claim 60, wherein the library of nucleic acid is a whole exome library.

62. The method of claim 60, wherein the library of nucleic acid is a library targeted toward rare abundance genes.

63. The method of any one of claims 59-62, wherein the sample of cfRNA is derived from blood, plasma, lymph, cerebrospinal fluid, amniotic fluid, urine, or stool.

64. A method of generating a targeted sequencing panel for sequencing of cell-free RNA, comprising: collecting a population of control liquid biopsies, each comprising cell-free RNA; performing sequencing on the cell-free RNA of the control liquid biopsies;identifying a set of rare abundance genes within the population of control liquid biopsies as defined by at least one or more of the following: their expression within a percentage of a population of control liquid biopsies or their expression level across the population of control liquid biopsies; and synthesizing a set of nucleic acid molecules that are for capturing or for amplifying the rare abundance genes to yield the targeted sequencing panel for sequencing of cell- free RNA.

65. The method of claim 64, wherein the set of rare abundance genes are defined by at least their expression within a percentage of a population of control liquid biopsies and their expression level across the population of control liquid biopsies.

66. The method of claim 64 or 65, wherein the yielded targeted sequencing panel for sequencing of cell-free RNA is the panel of nucleic acids of any one of claims 31-51.

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