Nucleic acid sequencing

Abstract

A nucleic acid sequencing method involving contacting a spatially defined target nucleic acid sequence with N labeled oligonucleotide probes and performing measurements, at least at the spatially defined site (1), at M time instances during ligation or hybridization to form M data sets. The M data sets are co-processed in order to identify a label or absence of any label at the spatially defined site (1). A sequenced based of the target nucleic acid sequence is then determined based on the identified label of the identified absence of any label. The time-resolved measurements conducted during the actual hybridization of the oligonucleotide probes to the target nucleic acid sequence or the ligation of oligonucleotide probes to anchor probes on the target nucleic acid sequence improves the speed and accuracy of the sequencing as compared to the state of the art sequencing methods.

1 - 18 . (canceled) 
     
     
         19 . A method comprising:
 a) contacting a sample with one or more probes;   b) performing measurements at at least two time points at a spatially defined site,   c) generating a combined data set from the at least two measurements, and   d) identifying a label or absence thereof by co-processing the combined data set, wherein identifying a label comprises detecting increased presence of the label at the spatially defined site over the at least two time points.   
     
     
         20 . The method of  claim 19 , wherein detecting increased presence of the label at the spatially defined site over the at least two time points is during one cycle. 
     
     
         21 . The method of  claim 19 , wherein detecting increased presence of the label at the spatially defined site over the at least two time points is during a plurality of cycles. 
     
     
         22 . The method of  claim 19 , wherein a single type of each of the one or more probes contacts the sample during a cycle. 
     
     
         23 . The method of  claim 22 , wherein the single type of each of the one or more probes has the same label during the cycle. 
     
     
         24 . The method of  claim 19 , wherein a) and b) are performed within one cycle of a plurality of cycles. 
     
     
         25 . The method of  claim 24 , wherein two or more types of the one or more probes contact the sample during one cycle of the plurality of cycles. 
     
     
         26 . The method of  claim 25 , wherein two or more types of the one or more probes have different labels in one cycle of the plurality of cycles. 
     
     
         27 . The method of  claim 19 , wherein the probes are oligonucleotide probes. 
     
     
         28 . The method according to  claim 27 , wherein the oligonucleotides probes contact target nucleic acid sequences in the sample. 
     
     
         29 . The method of  claim 27 , wherein the oligonucleotides probes are of different types and each type has a different nucleotide at a specific base position. 
     
     
         30 . The method of  claim 29 , comprising determining a sequence of a target nucleic acid sequence based on the identification of a label or absence thereof. 
     
     
         31 . The method according to  claim 19 , wherein the measurement comprises light measurements. 
     
     
         32 . The method according to  claim 31 , wherein the light measurements comprise measuring fluorescence, and the combined data set comprises a fluorescence data set. 
     
     
         33 . The method according to  claim 32 , wherein the light measurements comprise measuring fluorescence intensity in one or more fluorescence channels each for measuring a fluorescent label of the one or more probes. 
     
     
         34 . The method according to  claim 19 , wherein co-processing the combined data set comprises generating a combined data set of signal intensity projection. 
     
     
         35 . The method according to  claim 19 , wherein co-processing the combined data set comprises generating a combined data set of summing intensity values. 
     
     
         36 . The method according to  claim 19 , wherein co-processing the combined data comprises generating a combined data set of average intensity values. 
     
     
         37 . The method according to  claim 36 , wherein the another one or more probes have a higher affinity to the target nucleic acid sequence as compared to the one or more probes. 
     
     
         38 . The method of  claim 19 , wherein the steps are repeated to perform sequencing by ligation (SBL) or sequencing by ligation (SBH) of a rolling circle amplification product. 
     
     
         39 . The method of  claim 19 , wherein the method is performed in situ in a cell or tissue sample.