Detection and analysis of structural variations in genomes
Compositions and methods are provided for detection of dynamic loci in a genome, where such loci may comprise structural variations as a result of DNA recombination; DNA duplication, insertions, deletions, transpositions, and epigenetic changes. The methods may utilize microfluidic platforms and functionalized polymer matrices to allow determination of mechanisms of cell-type-specific, programmed genomic heterogeneity. The method and compositions allow determination of mechanisms of cell-type-specific, programmed genomic heterogeneity.
1. A method for analysis of structural variation within genomes; the method comprising:
isolating one or more cells, purified nuclei, or extracellular vesicles comprising nucleic acids from a biological sample;
providing a preformed polymer matrix;
embedding the one or more cells, purified nuclei, or extracellular vesicles comprising nucleic acids into the preformed polymer matrix to form individual pearls containing the one or more cells, purified nuclei, or extracellular vesicles;
lysing the one or more cells, purified nuclei, or extracellular vesicles in each of the pearls;
dissolving the polymer matrix of each pearl in a chaotropic dense salt solution;
fragmenting nucleic acids from the one or more cells, purified nuclei, or extracellular vesicles using a transposase;
adding barcodes and sequencing platform adaptors to the fragmented nucleic acids to obtain tagged nucleic acid fragments; and
sequencing the tagged nucleic acid fragments using modified sequencing primers.
2. The method of claim 1 , where nucleic acids are cross-linked prior to lysis.
3. The method of claim 1 , comprising non-destructive labeling of genomic DNA prior to analysis.
4. The method of claim 3 where the non-destructive labeling of genomic DNA prior to analysis is performed with a crosslinking reagent.
5. The method of claim 3 wherein the non-destructive labeling of genomic DNA prior to analysis is performed with a crosslinking reagent that generates chemically or physically reversible crosslinks.
6. The method of claim 1 , wherein the sequencing is performed with a high throughput platform.
7. The method of claim 1 wherein the step of embedding the one or more cells, purified nuclei, or extracellular vesicles comprising nucleic acids into the preformed polymer matrix to form individual pearls comprises;
mixing cells, oil and preformed polymer matrix in a microfluidic chamber.
8. The method of claim 1 , further comprising a step wherein the nucleic acids in pearls are subjected to digestion with an exonuclease or an endonuclease to digest a portion of the nucleic acids.
9. The method of claim 8 wherein the exonuclease is selected from the group consisting of Exonuclease V (RecBCD), Nuclease BAL-3, Thermolabile Exonuclease I, T7 Exonuclease, Nuclease P1, Exonuclease III, Exonuclease T, and T5 Exonuclease.
10. The method of claim 8 wherein the nucleic acids not digested with the exonuclease or endonuclease are circular DNA.
11. The method of claim 8 wherein the nucleic acids not digested with the exonuclease or endonuclease are RNA.
12. The method of claim 1 , wherein the nucleic acids in the transposase is selected from the group consisting of Tn5, Tn3, Tn7, Tn10, Mu, Mariner, SB, and RAG.
13. The method of claim 1 , wherein sequencing and index primers are used with a sequencing platform of interest.
14. The method of claim 13 wherein the index primers have modifications selected from introduction of Pyrene, Trimethoxystilbene, 2-Amino-deoxyadenosine, 5-Methyl-deoxycytidine, LNA, BNA, Aminoethyl-phenoxazine-deoxycytidine, C-5 Propynyl-deoxyuridine, C-5 Propynyl-deoxycytidine, or MGBs that increase melting temperature of the index primers compared to the index primers without the modifications.
15. The method of claim 1 , wherein the fragmented nucleic acids are obtained from circular nucleic acids.
16. The method of claim 15 , further comprising isolating the circular nucleic acids prior to the fragmenting of the circular nucleic acids.
17. The method of claim 8 , wherein a portion of non-digested nucleic acids are circular nucleic acids.
18. The method of claim 1 , wherein the one or more cells, purified nuclei, or extracellular vesicles comprising nucleic acids are injected into the preformed polymer matrix.
19. The method of claim 3 , wherein the non-destructive labeling of genomic DNA prior to analysis comprises a step of labeling the genomic DNA with a polynucleotide barcode.
20. The method of claim 3 , wherein the non-destructive labeling of genomic DNA prior to analysis comprises a step of labeling the genomic DNA with a fluorophore.