The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

The Invention is a method for ascertaining extremely long DNA sequence reads (kilobases or megabases) from polony-type DNA sequencers. Polony-type DNA sequencers (e.g., Illumina, Roche, and Life Technologies sequencers) typically give read lengths of only about 500 bp. The Invention can extend those read lengths by orders of magnitude.

The Invention is a method for ascertaining extremely long DNA sequence reads (kilobases or megabases) from polony-type DNA sequencers. Polony-type DNA sequencers (e.g., Illumina, Roche, and Life Technologies sequencers) typically give read lengths of only about 500 bp. The Invention can extend those read lengths by orders of magnitude.

Disclosed herein, inter alia, are novel methods pertaining to nucleic acid amplification and sequencing. Compositions for use in and produced by such methods are also provided.

Disclosed herein, inter alia, are novel methods pertaining to nucleic acid amplification and sequencing. Compositions for use in and produced by such methods are also provided.

Microscopy imaging that allow for multiple mRNAs to be resolved at a single cell level provides valuable information regarding transcript amount and localization, which is a crucial factor for understanding tissue heterogeneity, the molecular development and treatment of diseases. The current invention describes a method (Fly FISH) which combined the use of padlock oligonucleotides as fluorescence in situ hybridization (FISH) probes for detection and sequencing targeted portion of RNA or cDNA transcript at a cellular level with less limitation in the amount of transcripts and the length of the sequence that can be analyzed. Padlocks probes containing various barcodes in their core are utilized both as FISH probes and also to capture RNA portion that can be sequenced. The same barcodes can be used to selectively prime a rolling circle amplification and amplify a subset of transcripts coming from a specific region that have been tagged as of interest during the probing steps.

Microscopy imaging that allow for multiple mRNAs to be resolved at a single cell level provides valuable information regarding transcript amount and localization, which is a crucial factor for understanding tissue heterogeneity, the molecular development and treatment of diseases. The current invention describes a method (Fly FISH) which combined the use of padlock oligonucleotides as fluorescence in situ hybridization (FISH) probes for detection and sequencing targeted portion of RNA or cDNA transcript at a cellular level with less limitation in the amount of transcripts and the length of the sequence that can be analyzed. Padlocks probes containing various barcodes in their core are utilized both as FISH probes and also to capture RNA portion that can be sequenced. The same barcodes can be used to selectively prime a rolling circle amplification and amplify a subset of transcripts coming from a specific region that have been tagged as of interest during the probing steps.

The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.