Abstract: 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.

Abstract: The invention is directed to a method to obtain the spatial location and sequence information of a target sequence of at least one m-RNA strand on a tissue sample comprising the steps a. providing a linear probe, containing a) a binding region capable of binding to the at least one m-RNA strand and b) an anchor sequence comprising a UMI region located between a first and a second locator regions and c) a primer region; b. hybridizing the linear probe with its binding region to the m-RNA strand; c. complementing the linear probe using the m-RNA strand as template thereby obtaining a reversed transcribed c-DNA strand d. hybridizing a locator molecule with its 3? and 5? ends to the first and second locator regions thereby creating a gap corresponding to the length of the UMI of the linear probe e.

Abstract: The invention is directed to a method to simultaneously obtain both the spatial location and sequence information of a target sequence with a higher resolution than the known technologies. The method comprises steps to spatially localize the mRNA expressed on a tissue by the use of a hybrid circular/linear DNA probe with an UMI and—after several amplification steps, the obtaining sequence information by NGS.

Abstract: Microscopy imaging that allows for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (High Density-SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ localization and identification (by in situ sequencing or sequential fluorescence hybridization) of rolonies derived from rolling circle amplification of circular oligonucleotides and in vitro sequencing of target amplified RNA or DNA in combination with SUMI identification at a subcellular level with no optical diffraction limitation in the amount of amplified target information that can be analyzed per cell. Apart from amplified RNA or DNA, the High Density-SUMI-Seq method can also be applied using linear oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.

Abstract: The invention is directed to a method for obtaining the sequence information of a target sequence from a tissue comprising at least one RNA or c-DNA strand comprising two-fold RCA.

Abstract: Microscopy imaging that allow for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (High Density—SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ localization and identification (by in situ sequencing or sequential fluorescence hybridization) of rolonies derived from rolling circle amplification of circular oligonucleotides and in vitro sequencing of target captured RNA or DNA in combination with SUMI identification at a subcellular level with no optical diffraction limitation in the amount of captured target information that can be analyzed per cell. Apart from captured RNA or DNA, the High Density—SUMI-Seq method can also be applied using linear oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.

Abstract: The present invention is to provide a method for hybridization of a photo-responsive oligonucleotide to a nucleic acid by providing the nucleic acid with a complementary oligonucleotide, wherein the oligonucleotide functions as a starting point for a polymerase for nucleic acid synthesis characterized in that the photo-responsive oligonucleotide comprises at least two photo-responsive elements which change from a first to a second conformation upon irradiation with light thereby disabling or enabling the oligonucleotide hybridization. In addition to that, the current invention provides a method for spatially controlled oligonucleotide hybridization to specific sites by spatial illumination of areas of no interest, thus changing the oligonucleotide conformation to a non-binding state. The reversable hybridization of the oligonucleotide can be used for controlling several reactions such as rolling circle amplification and a sequencing reaction.

Abstract: Microscopy imaging that allow for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ sequencing and in vitro sequencing of rolonies derived from rolling circle amplification from padlock oligonucleotides targeting portion of RNA or cDNA transcript at a subcellular level with less limitation in the amount of transcripts and the length of the sequence that can be analyzed. Apart from padlocks oligonucleotides, the SUMI-Seq method can also be applied using circular oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.

Abstract: Cyclone Rolling Circle Amplification (CRCA) based next-generation sequencing (NGS) using a multiple primer rolling circle amplification (RCA) reaction is generated using two or more tandem primers from the same strand on different locations of library adaptor regions of double stranded enriched targeted polymerase chain reaction (PCR) library products. This process allows multiple initiation and syntheses of the RCA reaction by an enzyme on the same circular template molecule, which is beneficial since the two or more primers complement each other in generating uniform amplification of the target circle population. Also, a method for keeping DNA nanoballs (also known as rolonies) compact, or more compact, and for pre-priming rolonies before sequencing to eliminate the hybridization of a seqeuncing primer after seeding the rolonies, and a Rolonies rolling circle amplification (RCA) based next-generation sequencing (NGS) using a dual Rolonies primer approach named REPLI-Rolony.

Abstract: 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.

Abstract: Disclosed are compositions and methods for determining the nucleotide sequence of sequences of interest using paired-end sequencing. Dumbell circular templates can be generated and used in a rolling circle amplification reaction by ligating two hairpin adaptors on a double-stranded amplicon. Disclosed also are methods using double-stranded DNA, including both sense and antisense strands in a single circle to sequence, sequentially from the same concatemers.

Abstract: This invention relates in general to methods of sequencing multiple distinct and separate polynucleotide fragments and regions in a sequential order, such as on a flow cell surface. The invention provides methods that solve prior art problems with regard to sequential sequencing, and that provide advantages including low cost, shorter turn-around-time, high efficiency, and easy implementation.

Abstract: The present invention includes a rapid, selective, and accurate method of diagnosing a human subject with a triplet repeat genetic disorder of the FMR1 gene that leads to fragile X syndrome. The present invention also includes a rapid, selective, and accurate method of diagnosing a human subject at risk for developing a triplet repeat genetic disorder of the FMR1 gene that leads to fragile X syndrome, or at risk of passing such a disorder on to their progeny.

Abstract: The present invention encompasses a method of diagnosing chromosomal trisomy in a human subject. In one embodiment, the method comprises pyrosequencing at least one single nucleotide polymorphism on a chromosome being assessed for trisomy, where the SNP comprises two alleles.

Abstract: Disclosed are compositions and methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions.

Abstract: Disclosed are compositions and methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions.

Abstract: Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be ?29 DNA polymerase.

Abstract: Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be ?29 DNA polymerase.

Abstract: Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be &phgr;29 DNA polymerase.

Abstract: Disclosed are compositions and methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions.