Modifications to both hardware and enzymatic reactions used in single cell analyses such as but not limited to Seq-well that enable significant increases in the yield of transcripts per cell, portability and ease of use, increased scalability of the assay, and linkage of transcript information to other measurements made in the picowell arrays are disclosed.

Modifications to both hardware and enzymatic reactions used in single cell analyses such as but not limited to Seq-well that enable significant increases in the yield of transcripts per cell, portability and ease of use, increased scalability of the assay, and linkage of transcript information to other measurements made in the picowell arrays are disclosed.

Disclosed herein are methods of generating a subpool of oligonucleotides from a larger pool of oligonucleotides. A subpool of oligonucleotides can contain a portion of a gene of interest, and can be generated on a subject by subject basis on demand. Also disclosed herein are methods of processing a subpool of oligonucleotides to generate probes for sequencing. Also disclosed herein are systems and methods for using subpools of oligonucleotides generated from a larger pool for high-throughput sequencing.

Disclosed herein are methods of generating a subpool of oligonucleotides from a larger pool of oligonucleotides. A subpool of oligonucleotides can contain a portion of a gene of interest, and can be generated on a subject by subject basis on demand. Also disclosed herein are methods of processing a subpool of oligonucleotides to generate probes for sequencing. Also disclosed herein are systems and methods for using subpools of oligonucleotides generated from a larger pool for high-throughput sequencing.

Methods for the rapid amplification of extremely low quantity nucleic acids in a sample are provided. The disclosed methods are capable of amplifying less than 1 pg of DNA and/or RNA from a biological sample using a single tube and one-step or two-step preparation.

Methods for the rapid amplification of extremely low quantity nucleic acids in a sample are provided. The disclosed methods are capable of amplifying less than 1 pg of DNA and/or RNA from a biological sample using a single tube and one-step or two-step preparation.

The present disclosure relates to methods for amplifying nucleic acids that avoid problems associated with primer-dimer formation. The present methods are referred to herein as dimer avoided multiplex polymerase chain reaction (dam-PCR). The methods disclosed herein generally comprise the steps of reverse transcribing at least one first strand of DNA, for example cDNA from an RNA sample, wherein each first strand of DNA incorporates a reverse common primer binding site; selecting each first strand of DNA; synthesizing at least one second strand of DNA from each of the at least one first strand of DNA, wherein each second strand of DNA incorporates a forward common primer binding site; selecting each second strand of cDNA; and amplifying the DNA strands using common primers. Alternatively, the method may be performed using a gDNA template. The methods described herein, due to the selection of DNA strands and removal of unused primers prior to amplification, avoid primer-dimer formation and allow for greater sensitivity and efficiency compared with conventional multiplex PCR methods.

The present disclosure relates to methods for amplifying nucleic acids that avoid problems associated with primer-dimer formation. The present methods are referred to herein as dimer avoided multiplex polymerase chain reaction (dam-PCR). The methods disclosed herein generally comprise the steps of reverse transcribing at least one first strand of DNA, for example cDNA from an RNA sample, wherein each first strand of DNA incorporates a reverse common primer binding site; selecting each first strand of DNA; synthesizing at least one second strand of DNA from each of the at least one first strand of DNA, wherein each second strand of DNA incorporates a forward common primer binding site; selecting each second strand of cDNA; and amplifying the DNA strands using common primers. Alternatively, the method may be performed using a gDNA template. The methods described herein, due to the selection of DNA strands and removal of unused primers prior to amplification, avoid primer-dimer formation and allow for greater sensitivity and efficiency compared with conventional multiplex PCR methods.

Described herein are compositions and methods for the production and quantification of barcoded or unique molecular identifier (UMI)-labeled substrates. In one aspect, the substrate is a bead comprising a template oligonucleotide that is elongated by successive extension reactions to provide a bead with an oligonucleotide comprising a plurality of barcodes and conserved anchor regions. Methods are also described for quantifying the amount of template oligonucleotide loaded onto the substrate and the products of the extension reaction after each round and after the final extension.

Described herein are compositions and methods for the production and quantification of barcoded or unique molecular identifier (UMI)-labeled substrates. In one aspect, the substrate is a bead comprising a template oligonucleotide that is elongated by successive extension reactions to provide a bead with an oligonucleotide comprising a plurality of barcodes and conserved anchor regions. Methods are also described for quantifying the amount of template oligonucleotide loaded onto the substrate and the products of the extension reaction after each round and after the final extension.