The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single-stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, lncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, lncRNA's splice-site variants, translocations, and miRNA.

Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.

Provided herein are compositions and methods for generating libraries for high throughput sequencing without using ligation, and methods of using same.

The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors. The double-stranded splint adaptors (200) can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor (200) comprises a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor (200) having a double-stranded region and two flanking single-stranded regions. The second splint strand (400) carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence and/or an index sequence.

The present disclosure provides compositions comprising nucleic acid single-stranded splint strands, including kits, and methods that employ the single-stranded splint strands. The single-stranded splint strands can hybridize to portions of linear library molecules to form circularized library-splint complexes having a nick, where the nick can be ligated to form covalently closed circular molecules which can be subjected to downstream amplification and sequencing workflows.

The present disclosure provides systems, methods, and apparatus for preparing biological samples (e.g., plasma) for sequencing (e.g., DNA sequencing, e.g., third generation sequencing). Moreover, the present disclosure provides various systems, methods, and apparatus that employ this sample preparation technology in the identification of biomarkers for detection of a disease or condition. For example, in certain embodiments, the biological sample preparation method includes capturing fragments of cell free DNA (cfDNA) with capture probes, converting the captured DNA fragments into circular DNA, and amplifying the circular DNA by performing rolling circle amplification (RCA). In particular, it is presently found that by performing this sample preparation method, it is possible to more successfully distinguish true alterations (e.g., aberrant methylation status and/or genomic mutations) from technical/sequencing artifacts.

A method for producing pancreatic endocrine cells, including introducing (A), (B), (C), or (D) into somatic cells: (A) mutated GLIS1 gene having ≥85%-sequence-identity to base sequence of SEQ ID NO: 1 or 2 or gene product(s) thereof, Neurogenin3 gene or gene product(s) thereof, Pdx1 gene or gene product(s) thereof, and MafA gene or gene product(s) thereof, (B) mutated GLIS1 gene having ≥85%-sequence-identity to base sequence of SEQ ID NO: 1 or 2 or gene product(s) thereof, Neurogenin3 gene or gene product(s) thereof, and Pdx1 gene or gene product(s) thereof. (C) GLIS1 gene or gene product(s) thereof, Neurogenin3 gene or gene product(s) thereof, Pdx1 gene or gene product(s) thereof, and MafA gene or gene product(s) thereof, and (D) mutated GLIS1 gene having ≥85%-sequence-identity to base sequence of SEQ ID NO: 1 or 2 or gene product(s) thereof, Neurogenin3 gene or gene product(s) thereof, and MafA gene or gene product(s) thereof.

Methods of labeling or barcoding molecules within one or more portions of a plurality of cells are provided. Kits and systems for labeling or barcoding molecules within one or more portions of a plurality of cells are also provided. The methods, kits, and systems may utilize photo-controlled adapter sequences, nucleic acids tags, and/or linkers.

The present disclosure provides nucleic acid molecules, compositions, and kits comprising the same, and methods for producing templated assembly products.

A method of characterizing candidate agents including steps of (a) providing a library of candidate agents attached to nucleic acid tags; (b) contacting the library with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed; (c) contacting the array with a screening agent, wherein one or more candidate agents in the array react with the screening agent; (d) detecting the array to determine that at least one candidate agent in the array reacts with the screening agent; (e) sequencing the nucleic acid tag to determine the tag sequences attached to candidate agents in the array; and (f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent.