Methods and compositions for sequencing library preparation

Abstract

Provided herein are methods of proximity ligation and compositions for use in such methods.Also provided herein are embodiments for evaluating single-cell nucleic acid conformation or determining single-cell nucleic acid sequence or phasing information.Conformation-preserved or conformation-reconstructed nucleic acid samples can be fragmented and distributed into aliquots with aliquot-identifying sequence segments added so that paired ends can be assigned to partition origin or cell origin upon analysis of paired-end libraries generated from the samples.Thus, cell-specific changes in sequence and three-dimensional nucleic acid configuration can be determined.

Claims

1. 1. A method for processing nucleic acids, the method comprising: (a) obtaining a stabilized sample comprising nucleic acid molecules complexed to at least one nucleic acid binding protein; (b) cleaving the nucleic acid molecule into a plurality of segments comprising at least a first segment and a second segment, wherein the cleaving is accomplished by a transposase; (c) attaching a first recombinase site to the first segment and the second segment; (d) contacting the first segment and the second segment with a linker comprising a second recombinase site in the presence of a recombinase, thereby producing a linked nucleic acid comprising a first sequence from the first segment, a linker sequence from the linker, and a second sequence from the second segment; A method comprising:

2. The method of claim 1, wherein the transposase is a Tn5 transposase.

3. 2. The method of claim 1, wherein the second recombinase site comprises two recombinase sites oriented as direct repeats.

4. 2. The method of claim 1, wherein the recombinase is an integrase, and optionally the integrase is PhiC31 integrase, Bxb1 integrase, or a combination thereof.

5. The method of claim 1, further comprising the step of circularizing the bound nucleic acid by ligating the 5' end of the bound nucleic acid to the 3' end of the bound nucleic acid.

6. The method of claim 1, further comprising a step of sequencing at least a portion of the bound nucleic acid, optionally wherein the step of sequencing comprises sequencing at least a portion of the first sequence and at least a portion of the second sequence.

7. The method of claim 6, further comprising a step of mapping at least a portion of the first sequence and at least a portion of the second sequence to a genome.

8. The method of claim 6, further comprising a step of performing three-dimensional genomic analysis using information from the sequencing step.

9. The method of claim 1, wherein the stabilized sample is a cross-linked sample.

10. The method of claim 1, wherein the nucleic acid binding protein comprises chromatin or a component thereof.

11. The method of claim 1, wherein the first recombinase site and the second recombinase site comprise attP and attB integrase sites or transposase mosaic ends.

12. The method of claim 1, wherein the linker sequence includes a barcode sequence, and optionally, the barcode sequence indicates a partition origin.

13. The method of claim 1, wherein the method is completed in four hours or less.

14. The method of claim 1, wherein the stabilized sample contains 50,000 cells or less.

15. The method of claim 1, wherein the stabilized sample comprises stabilized nuclei, optionally comprising less than 50,000 nuclei.

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