Methods and systems for sample processing or analysis

Abstract

The present disclosure provides methods and systems for detecting nucleic acid sequences in a biological sample having a three-dimensional matrix. The present disclosure also provides methods and systems for processing a biological sample for use in nucleic acid sequence detection.

1 . A method for identification of a nucleic acid sequence in a biological sample, comprising:
 (a) providing said biological sample comprising a ribonucleic acid (RNA) molecule hybridized to a deoxyribonucleic acid molecule (DNA) in a three-dimensional (3D) matrix, wherein said RNA molecule comprises said nucleic acid sequence, and wherein said DNA molecule comprises an additional nucleic acid sequence that is a reverse complement of said nucleic acid sequence;   (b) degrading or digesting at least a portion of said RNA molecule hybridized to said DNA molecule, wherein said at least said portion of said RNA is degraded or digested (i) using a reverse transcriptase or (ii) non-enzymatically; and   (c) detecting said additional nucleic acid sequence, thereby identifying said nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , wherein said DNA molecule is immobilized to said 3D matrix. 
     
     
         3 . The method of  claim 2 , wherein said DNA molecule comprises a functional moiety, and wherein said DNA molecule is immobilized to said 3D matrix via said functional moiety. 
     
     
         4 . The method of  claim 1 , wherein said RNA molecule is immobilized to said 3D matrix. 
     
     
         5 . The method of  claim 4 , wherein said RNA molecule comprises a functional moiety, and wherein said RNA molecule is immobilized to said 3D matrix via said functional moiety. 
     
     
         6 . The method of  claim 1 , wherein (c) comprises contacting said DNA molecule with a probe. 
     
     
         7 . The method of  claim 6 , wherein said probe comprises a functional moiety, and wherein said probe is immobilized to said 3D matrix via said functional moiety. 
     
     
         8 . The method of  claim 6 , wherein said probe is a padlock probe, wherein said padlock probe comprises 5′ and 3′ terminal regions complementary to said DNA molecule, and wherein said 5′ and 3′ terminal regions of said padlock probe are hybridized to said DNA molecule. 
     
     
         9 . The method of  claim 8 , further comprising circularizing said padlock probe by ligating two ends of said padlock probe together, to yield a circularized padlock probe. 
     
     
         10 . The method of  claim 9 , wherein said two ends of said padlock probe are contiguous. 
     
     
         11 . The method of  claim 9 , wherein said two ends of said padlock probe are separated by a gap region comprising at least one nucleotide. 
     
     
         12 . The method of  claim 11 , wherein said gap region comprises from 2 to 500 nucleotides. 
     
     
         13 . The method of  claim 11 , further comprising filling said gap region by incorporating at least one nucleotide in an extension reaction. 
     
     
         14 . The method of  claim 11 , further comprising filling said gap region by at least one additional nucleotide or an oligonucleotide sequence. 
     
     
         15 . The method of  claim 9 , further comprising subjecting said circularized padlock probe to rolling circle amplification (RCA) to generate a nucleic acid molecule comprising a nucleic acid sequence of said circularized padlock probe, which nucleic acid molecule comprises a nucleic acid sequence corresponding to said nucleic acid sequence of said RNA molecule. 
     
     
         16 . The method of  claim 15 , further comprising detecting said nucleic acid sequence of said nucleic acid molecule, thereby identifying said nucleic acid sequence of said RNA molecule. 
     
     
         17 . The method of  claim 1 , further comprising, prior to (a), reverse transcribing said RNA molecule to generate said DNA molecule hybridized to said RNA molecule in said biological sample. 
     
     
         18 . The method of  claim 17 , wherein said RNA molecule is reverse transcribed using an additional reverse transcriptase. 
     
     
         19 . The method of  claim 17 , further comprising, prior to (a), hybridizing a reverse transcription primer to said RNA molecule, wherein said reverse transcription primer comprises a functional moiety, and wherein said DNA molecule is immobilized to said 3D matrix via said function moiety. 
     
     
         20 . The method of  claim 17 , wherein (b) is performed under a first set of conditions and reverse transcribing said RNA molecule is performed under a second set of conditions, wherein said first set of conditions is different than said second set of conditions. 
     
     
         21 - 30 . (canceled)