Single cell analysis of transposase accessible chromatin

Abstract

Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.

1 . A method of generating barcoded nucleic acids, comprising:
 (a) obtaining a biological particle comprising a template deoxyribonucleic acid (DNA) molecule and a template ribonucleic acid (RNA) molecule;   (b) generating a template DNA fragment derived from said template DNA molecule in said biological particle with the aid of a transposase molecule and a transposon end oligonucleotide molecule;   (c) partitioning said biological particle into a partition such that said partition comprises (i) said biological particle comprising said template DNA fragment and said template RNA molecule; (ii) a first nucleic acid barcode molecule comprising a first barcode sequence; and (iii) a second nucleic acid barcode molecule comprising a second barcode sequence; and   (d) synthesizing a barcoded DNA fragment using flail said first nucleic acid barcode molecule and said template DNA fragment; and   (e) synthesizing a barcoded complementary DNA (cDNA) molecule using said template RNA molecule and said second nucleic acid barcode molecule.   
     
     
         2 . The method of  claim 1 , wherein said second nucleic acid barcode molecule further comprises a capture sequence. 
     
     
         3 . The method of  claim 1 , wherein said biological particle is a single nucleus. 
     
     
         4 . The method of  claim 1 , wherein said biological particle is a single cell. 
     
     
         5 . The method of  claim 1 , wherein said first barcode sequence and said second barcode sequence are the same. 
     
     
         6 . The method of  claim 1 , wherein said first barcode sequence and said second barcode sequence are different. 
     
     
         7 . The method of  claim 2 , wherein said capture sequence is an oligo(dT) sequence. 
     
     
         8 . The method of  claim 1 , wherein (e) comprises synthesizing a first barcoded cDNA strand and synthesizing a second barcoded cDNA strand. 
     
     
         9 . The method of  claim 8 , wherein said partition further comprises a template switching oligonucleotide and wherein said second barcoded cDNA strand is generated with the aid of said template switching oligonucleotide. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein said template DNA fragment and said template RNA molecule are released from said biological particle. 
     
     
         12 . The method of  claim 1 , wherein, in (c),
 said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are attached to a bead.   
     
     
         13 . The method of  claim 12 , wherein said bead is a gel bead. 
     
     
         14 . The method of  claim 12 , wherein said first nucleic acid barcode molecule and said second nucleic acid barcode molecule are releasably attached to said bead. 
     
     
         15 . The method of  claim 14 , further comprising releasing first nucleic acid barcode molecule and said second nucleic acid barcode molecule from said bead. 
     
     
         16 . The method of  claim 14 , wherein said bead comprises a polymer and said bead is depolymerized to release said first nucleic acid barcode molecule and said second nucleic acid barcode molecule from said bead. 
     
     
         17 . The method of  claim 16  wherein said partition comprises a reducing agent atoll that depolymerizes said bead. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein said partition is a droplet. 
     
     
         20 . The method of  claim 1 , wherein said partition is a well. 
     
     
         21 . The method of  claim 1 , wherein said first nucleic acid barcode molecule is attached to a gel bead, and wherein said second nucleic acid barcode molecule is attached to a magnetic bead embedded within said gel bead. 
     
     
         22 . The method of  claim 21 , wherein said first nucleic acid barcode molecule is releasably attached to said gel bead. 
     
     
         23 . The method of  claim 22 , further comprising releasing said first nucleic acid barcode molecule and said magnetic bead from said gel bead. 
     
     
         24 . The method of  claim 23 , wherein said gel bead comprises a polymer and is depolymerized to release said first nucleic acid barcode molecule and said magnetic bead from said gel bead. 
     
     
         25 . The method of  claim 24  wherein said partition comprises a reducing agent that depolymerizes said gel bead. 
     
     
         26 . The method of  claim 1 , wherein said barcoded DNA fragment and said barcoded cDNA molecule are released from said partition. 
     
     
         27 . The method of  claim 1 , further comprising: sequencing said barcoded DNA fragment, or derivative thereof; sequencing said barcoded cDNA molecule, or derivative thereof; or sequencing said barcoded DNA fragment and said barcoded cDNA molecule, or derivatives thereof. 
     
     
         28 . The method of  claim 1 , wherein said biological particle comprises chromatin and wherein said barcoded DNA fragment, or derivative thereof, is indicative of an accessible region of said chromatin of said biological particle. 
     
     
         29 . The method of  claim 2 , wherein said template RNA molecule is attached to said second nucleic acid barcode molecule and wherein, prior to (e), said template RNA molecule attached to said second nucleic acid barcode molecule is released from said partition. 
     
     
         30 . The method of  claim 29 , wherein said template RNA molecule is attached to said second nucleic acid barcode molecule by hybridization of said template RNA molecule with said capture sequence of said second nucleic acid barcode molecule. 
     
     
         31 . The method of  claim 1 , wherein said first nucleic acid barcode molecule is attached to a first bead, and wherein said second nucleic acid barcode molecule is attached to a second bead. 
     
     
         32 . The method of  claim 1 , wherein (d) and (e) occur in said partition.