10x Genomics, Inc.

Malte Kühnemund

Toon Verheyen

Jorge Iván Hernández Neuta

Mats Nilsson

Tomasz Krzywkowski

Patrick Marks

Christina Galonska

Xiaoyan Qian

Jessica Östlin

Thomas Hauling

Ariel Royall

In some aspects disclosed herein are methods and compositions for detecting analytes such as a plurality of nucleic acid molecules in a biological sample, said method comprising generating and analyzing a first detectable signal and a second detectable signal corresponding to a first target sequence and a second target sequence, respectively. The first and second target sequences can be present and/or expressed at different levels in the biological sample.

Multiple readout signals for analyzing a sample

In some aspects, the present disclosure relates to methods for reducing the detection of false positive ligation events. In some aspects, the method comprises use of a double split (or 'split split') probe. The methods herein have particular applicability in reducing the detection of false positive ligation events when using ligases that have high ligation efficiency but low specificity (e.g., SplintR® ligase). Also provided are kits comprising probes for use in such methods.

Probes comprising a split barcode region and methods of use

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.

Methods and compositions for synchronizing reactions in situ

Provided herein are methods, compositions, and kits for the spatial analysis of target nucleic acids, or complements thereof, by their 5' end.

Methods, compositions, and kits for determining the location of an analyte in a biological sample

In some aspects disclosed herein are methods and compositions for detecting a target nucleic acid molecule, said method comprising performing a linear oligo hybridization chain reaction (LO-HCR) to generate a polymeric product, and detecting the polymeric product, thereby detecting the target nucleic acid molecule.

Methods of detecting an analyte

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5' to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

RNA templated ligation

The present application provides compositions, systems, and methods for encoding and decoding nucleic acid molecules.

Sequential decoding of nucleic acids

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

Circularizable probes for in situ analysis

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5' to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5&#x2032; flap containing one or more nucleotides at its 3&#x2032; end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

The present application provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule and a plurality of labelled detection probes which yield signals which make up the signal code sequence. In particular, the invention provides a sequential barcoding and decoding scheme which utilises a sequencing-by-hybridisation (SBH) strategy to sequence and decode a nucleotide barcode sequence, and to differentiate the nucleotide barcode sequence from other nucleotide barcode sequences. In an extension of the method, the application also provides a new coding scheme for providing a target nucleic acid with a detectable 'colour' (or similar signal)-based code.

Method of detecting target nucleic acid molecules

A nucleic acid sequencing method involving contacting a spatially defined target nucleic acid sequence with N labeled oligonucleotide probes and performing measurements, at least at the spatially defined site (1), at M time instances during ligation or hybridization to form M data sets. The M data sets are co-processed in order to identify a label or absence of any label at the spatially defined site (1). A sequenced based of the target nucleic acid sequence is then determined based on the identified label of the identified absence of any label. The time-resolved measurements conducted during the actual hybridization of the oligonucleotide probes to the target nucleic acid sequence or the ligation of oligonucleotide probes to anchor probes on the target nucleic acid sequence improves the speed and accuracy of the sequencing as compared to the state of the art sequencing methods.

Growing community of inventors

Stockholm, Sweden

Malte Kühnemund