Sequencing and analysis of exosome associated nucleic acids
The invention provides a series of steps that prepare nucleic acids (RNA and/or DNA) isolated from extracellular vesicles for sequencing. This enables a wide diversity of RNAs and/or DNAs, to be efficiently detected. These can then be used to identify various attributes such as gene expression, alternative splicing, and the detection of both somatic and germline mutations including single nucleotide variants (SNV) and structural variations (insertions/deletions, fusions, inversions).
1. A method for sequencing at least one long non-ribosomal microvesicular RNA transcript from a biological sample, wherein the long non-ribosomal microvesicular RNA transcripts comprises more than 200 nucleotides and comprises long non-coding RNA, mRNA, circular RNA or any combination thereof,
the method comprising:
(a) contacting the biological sample with a solid capture surface comprising an affinity membrane that binds microvesicles to retain extracellular vesicles comprising long non-ribosomal microvesicular RNA transcripts from the biological sample on or in the capture surface;
(b) contacting the capture surface with a lysis reagent while the extracellular vesicles are on or in the capture surface, thereby releasing the long non-ribosomal microvesicular RNA transcripts from the capture surface and producing a homogenate;
(c) extracting the long non-ribosomal microvesicular RNA transcripts from the homogenate;
(d) reverse transcribing the extracted long non-ribosomal microvesicular RNA transcripts into cDNA;
(e) constructing a double-stranded DNA library from the reverse-transcribed cDNA;
(f) selectively removing ribosomal RNA or RNA sequences from the double-stranded DNA library;
(g) selectively enriching for nucleic acid sequences from the double-stranded DNA library; and
(h) sequencing the selectively enriched nucleic acid sequences from the double-stranded DNA library, thereby sequencing the long non-ribosomal microvesicular RNA transcripts.
2. The method of claim 1 , further comprising before or after step (c), pretreating the homogenate or the extracted long microvesicular non-ribosomal RNA transcripts with DNase, wherein the DNase is DNase I or modified DNase I.
3. The method of claim 1 , wherein the step of selectively removing ribosomal DNA or RNA sequences from the double-stranded DNA library comprises using enzymatic reagents, RNase H, restriction enzyme digest, hybridization-based biotinylated probe enrichment and streptavidin conjugated paramagnetic beads, or any combination thereof.
4. The method of claim 1 , wherein the step of selectively enriching for nucleic acid sequences from the double-stranded DNA library comprises using PCR-based approaches, complementary oligonucleotides, hybridization-based biotinylated probe enrichment and streptavidin conjugated paramagnetic beads, or any combination thereof.
5. The method of claim 1 , wherein the long non-ribosomal microvesicular RNA transcripts comprises more than 300 nucleotides, or more than 500 nucleotides.
6. The method of claim 1 , wherein the biological sample has a volume of about 0.5 mL to about 20 mL, about 0.5 mL to about 10 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 4 mL, or about 0.5 mL to about 2 mL.
7. The method of claim 1 , wherein the biological sample is selected from the group consisting of blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof.
8. The method of claim 1 , wherein the solid capture surface comprises a membrane or a bead.
9. The method of claim 1 , wherein the solid capture surface comprises more than one membrane, at least two membranes, at least three membranes.
10. The method of claim 1 , wherein the solid capture surface is magnetic.
11. The method of claim 1 , wherein the solid capture surface comprises a bead which is a positively charged ion exchange (IEX) bead, a negatively charged IEX bead, a high capacity IEX bead, a strong ferromagnetic high capacity IEX bead, a strong ferromagnetic high capacity iron oxide-containing polymer IEX bead or any combination thereof.
12. The method of claim 1 , wherein the solid capture surface is functionalized with quaternary ammonium, quaternary amine, sulfate, sulfonate, tertiary amine, or any combination thereof.
13. The method of claim 1 , wherein the solid capture surface comprises an IEX bead having a high ratio of bead charge to exposed surface.
14. The method of claim 1 , wherein step (c) further comprises adding protein precipitation buffer to the homogenate prior to extraction of the long non-ribosomal microvesicular RNA transcripts.
15. The method of claim 1 , wherein step (c) further comprises performing an enzymatic digestion, performing a proteinase digestion, performing a digestion using DNase, performing a digestion using RNase or any combination thereof.
16. The method of claim 1 , wherein step (c) further comprises adding a protein precipitation buffer, wherein the protein precipitation buffer comprises a transition metal ion, a buffering agent, or both a transition metal ion and a buffering agent.
17. The method of claim 1 , wherein step (a) further comprises filtering the biological sample.
18. The method of claim 1 , wherein step (b) further comprises washing the capture surface after contacting the biological sample with the capture surface.
19. The method of claim 1 , wherein step (c) comprises the addition of isopropanol, sodium acetate, glycogen or any combination thereof.
20. The method of claim 1 , wherein after step (e), the double-stranded DNA library is amplified.