METHODS AND SYSTEMS FOR REDUCING PARTICLE AGGREGATION
The present disclosure provides methods, systems, compositions, and kits for reducing particle aggregation and improving sequencing quality. A method for processing particles may comprise the use of a first buffer solution or a second buffer solution. A method for processing particles may comprise the use of a single-strand binding moiety such as a single-strand binding protein.
1 - 98 . (canceled)
99 . A method for processing a plurality of particles, comprising:
(a) providing a first particle of said plurality of particles comprising a first nucleic acid molecule immobilized thereto, wherein said first nucleic acid molecule comprises a first single-stranded portion;
(b) contacting said first nucleic acid molecule with a first single-stranded binding moiety under conditions sufficient to couple said first single-stranded binding moiety to said first single-stranded portion of said first nucleic acid molecule to yield a first treated particle comprising a first blocked nucleic acid molecule immobilized thereto, wherein said first blocked nucleic acid molecule comprises said first nucleic acid molecule coupled to said first single-stranded binding moiety; and
(c) providing said first treated particle in a solution comprising a second treated particle, wherein said second treated particle comprises a second nucleic acid molecule comprising a second single-stranded portion coupled to a second single-stranded binding moiety.
100 . The method of claim 99 , wherein said first nucleic acid molecule comprises a sequence of a sample nucleic acid molecule or a complement thereof, a priming sequence or a complement thereof, or a combination thereof.
101 . The method of claim 99 , wherein said first nucleic acid molecule comprises a barcode sequence or a unique molecular identifier sequence.
102 . The method of claim 99 , wherein (b) comprises providing a reaction mixture comprising said first single-stranded binding moiety.
103 . The method of claim 102 , wherein said reaction mixture comprises spermine.
104 . The method of claim 102 , wherein said reaction mixture comprises cobalt hexammine.
105 . The method of claim 99 , wherein said first single-stranded binding moiety comprises a single-stranded binding (SSB) protein.
106 . The method of claim 99 , wherein said first single-stranded binding moiety comprises a third nucleic acid molecule.
107 . The method of claim 106 , wherein said first single-stranded binding moiety comprises a loop structure.
108 . The method of claim 99 , wherein said first particle comprises a first plurality of nucleic acid molecules immobilized thereto, wherein said first plurality of nucleic acid molecules comprises said first nucleic acid molecule, and wherein each of said first plurality of nucleic acid molecules has at least partial sequence identity to a first nucleic acid sequence.
109 . The method of claim 99 , further comprising immobilizing said first particle to a substrate.
110 . The method of claim 109 wherein said substrate comprises said solution.
111 . The method of claim 99 , wherein (b) comprises contacting said plurality of particles with a plurality of single-stranded binding moieties under conditions sufficient to couple said single-stranded binding moieties of said plurality of single-stranded binding moieties to single-stranded portions of nucleic acid molecules immobilized to particles of said plurality of particles to yield a plurality of treated particles comprising said first treated particle and said second treated particle.
112 . The method of claim 111 , wherein, subsequent to (b), particle aggregates comprising two or more treated particles of said plurality of treated particles and having a dimension of at least about 1 micrometer (µm) are absent from said plurality of treated particles.
113 . The method of claim 111 , wherein, subsequent to (b), no more than 1% of treated particles of said plurality of treated particles are included in a particle aggregate comprising two or more treated particles of said plurality of treated particles.
114 . The method of claim 111 , further comprising immobilizing said plurality of treated particles to a substrate.
115 . The method of claim 99 , further comprising, prior to (a), denaturing a doublestranded portion of said first nucleic acid molecule to yield said first single-stranded portion.
116 . The method of claim 99 , further comprising sequencing said first single-stranded portion of said first nucleic acid molecule, or a portion thereof.
117 . A method for processing a plurality of particles, comprising:
(a) providing said plurality of particles, wherein each particle of at least a subset of said plurality of particles comprises a nucleic acid molecule of a plurality of nucleic acid molecules immobilized thereto, wherein each nucleic acid molecule of at least said subset of said plurality of nucleic acid molecules comprises a single-stranded portion; and
(b) contacting said plurality of particles with a plurality of single-stranded binding moieties under conditions sufficient for single-stranded binding moieties of said plurality of single-stranded binding moieties to couple to single-stranded portions of nucleic acid molecules of said at least said subset of said plurality of nucleic acid molecules,
wherein, subsequent to (b), said plurality of particles are included in a solution, and
wherein no more than 1% of particles of said plurality of particles are included in a particle aggregate comprising two or more particles of said plurality of particles.
118 . The method of claim 117 , wherein said plurality of single-stranded binding moieties comprises a plurality of single-stranded binding (SSB) proteins.