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: DNA sequencing by sequential addition/incorporation of non 3’capped and fluorescently labeled nucleotides. Each incorporation of individual nucleotides (A, or T or G or C) are separated by a wash. After all incorporation using all four bases, the substrate is imaged then the dyes are cleaved, and the following cycle of incorporations, washes, imaging and cleave is resumed.

Abstract: The invention is directed to a method for detecting RNA, DNA or protein target sequences by a) Hybridizing a library of probes having the general formula (I) P—(CL-D)x??(I) ?With P: probes having at least 10 nucleotides or amino acids CL: cleavable linker D: fluorescent dye X: integer between 1 and 5 ?to RNA, DNA or protein target sequences wherein the library comprises probes P having different sequences of nucleotides or amino acids and cleavable linkers CL of different groups which are cleavable with different means b) Removing unhybridized probes and detecting the hybridized probes via the fluorophores D by a first image c) Cleaving sequentially by different means each group of chemical linkers CL from the hybridized probes; removing the thus cleaved fluorophores D and detecting the remaining hybridized probes via their fluorophores D by a second image d) Detecting the removed fluorophores D by comparing the first and second image.

Abstract: The invention is directed to a method to obtain the spatial location and sequence information of an m-RNA target sequence on a tissue sample comprising providing a solid surface, attaching anchor molecules, binding scaffolding molecules, incorporating adenine, guanine, cytosine and thymine, incorporating thymine to the anchor molecules, removing the scaffolding, providing a tissue sample, reverser transcrining to create c-DNA, removing the c-DNA and obtaining the sequence information of the c-DNA.

Abstract: The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5? and 3? ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f.

Abstract: A microfabricated droplet dispensing structure is described, which may include a MEMS microfluidic fluidic valve, configured to open and close a microfluidic channel. The opening and closing of the valve may separate a target biological particle containing genomic material, and a bead from a sample stream, and direct these two particle into a single droplet formed at the edge of the substrate. The droplet may then be encased in a sheath flow of an immiscible fluid, and provided to a downstream workflow.

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: A microfabricated droplet dispensing structure is described, which may include a MEMS microfluidic fluidic valve, configured to open and close a microfluidic channel. The opening and closing of the valve may separate a target biological particle containing genomic material, and a bead from a sample stream, and direct these two particle into a single droplet formed at the edge of the substrate. The droplet may then be encased in a sheath flow of an immiscible fluid, and provided to a sequencing module. The sequencing module may sequence the genomic material and/or an identifying barcode attached to the bead.

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: A method is described for the reproducible quantification of biomarker expression, including biomarker expression in a tissue sample. Methods and systems are described whereby reproducible scores for biomarker expression are obtained independent of instrument, its location, or operator.

Abstract: The invention discloses a novel hair holding device that facilitates attachment of a section of hair into a ponytail while also reducing the difficulty with which it can be tighten or removed. The device is a band made of a flexible non-slippery material, preferably silicone, containing equidistant apertures at each extremity. These apertures are used to insert a dumbbell-shape attachment component in order to fasten and tighten hair in a ponytail or other hairstyles. The attachment portion of the device also is hollow allowing for the insertion of decorative signs or accessories. The device can also be converted into a fashionable bracelet and more rugged versions can be used to bundle cords or other miscellaneous objects.

Abstract: A centrifugable liquid vessel integrating a magnet in its lid and used for the separation of magnetic beads in suspension in a liquid. The device allows the separation of the magnetic beads and their efficient transfer or wash in the same or in a new vessel without the use of a pipetting device. The segregation, wash and transfer of the beads without aspiration significantly reduce the risks of contamination and maximize the magnetic beads recovery.

Abstract: Methods and apparatus for standardizing quantitative measurements from a microscope system. The process includes a calibration procedure whereby an image of a calibration slide is obtained through the optics of the microscope system. The calibration slide produces a standard response, which can be used to determine a machine intrinsic factor for the particular system. The machine intrinsic factor can be stored for later reference. In use, images are acquired of a target sample and of the excitation light source. The excitation light source sample is obtained using a calibration instrument configured to sample intensity. The calibration instrument has an associated correction factor to compensate its performance to a universally standardized calibration instrument. The machine intrinsic factor, sampled intensity, and calibration instrument correction factor are usable to compensate a quantitative measurement of the target sample in order to normalize the results for comparison with other microscope systems.