DNA Barcode Compositions and Methods of In Situ Identification in a Microfluidic Device

A method of in-situ identification of one or more capture objects within a microfluidic device, said method comprising:

• disposing a single capture object of said one or more capture objects within an isolation region of each of one or more sequestration pens located within an enclosure of said microfluidic device, wherein each capture object comprises a plurality of capture oligonucleotides, and wherein each capture oligonucleotide of said plurality comprises:
• a priming sequence;
• a capture sequence;
• and
• a barcode sequence, wherein said barcode sequence comprises three or more cassetable oligonucleotide sequences, each cassetable oligonucleotide sequence being non-identical to the other cassetable oligonucleotide sequences of said barcode sequence;
• flowing a first reagent solution comprising a first set of hybridization probes into a flow region within said enclosure of said microfluidic device, wherein said flow region is fluidically connected to each of said one or more sequestration pens, and wherein each hybridization probe of said first set comprises:
• an oligonucleotide sequence complementary to a cassetable oligonucleotide sequence comprised by any of said barcode sequences of any of said capture oligonucleotides of any of said one or more capture objects, wherein said complementary oligonucleotide sequence of each hybridization probe in said first set is non-identical to every other complementary oligonucleotide sequence of said hybridization probes in said first set;
• and
• a fluorescent label selected from a set of spectrally distinguishable fluorescent labels, wherein said fluorescent label of each hybridization probe in said first set is different from the fluorescent label of every other hybridization probe in said first set of hybridization probes;
• hybridizing said hybridization probes of said first set to corresponding cassetable oligo-nucleotide sequences in any of said barcode sequences of any of said capture oligonucleotides of any of said one or more capture objects;
• detecting, for each hybridization probe of said first set of hybridization probes, a corresponding fluorescent signal associated with any of said one or more capture objects;
• and
• generating a record, for each capture object disposed within one of said one or more sequestration pens, comprising (i) a location of said sequestration pen within said enclosure of said microfluidic device, and (ii) an association or non-association of said corresponding fluorescent signal of each hybridization probe of said first set of hybridization probes with said capture object, wherein said record of associations and non-associations constitute a barcode which links said capture object with said sequestration pen.