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High-throughput nucleotide library sequencing

A technology of nucleotides and polynucleotides, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA preparation, etc., and can solve problems such as unrelated

Active Publication Date: 2017-08-01
ABVITRO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Surprisingly, by immunosequencing using human antibody repertoires, no correlation has been found between antibody framework expression frequency and antibody activation potential in response to immune challenge

Method used

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  • High-throughput nucleotide library sequencing
  • High-throughput nucleotide library sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1a

[0342] Example 1a - Protocol for preparing cells for emulsion-based large-scale high-throughput single-cell polynucleotide sequencing.

[0343] Obtain the cell population of interest. These include total PBMC, sorted cells, antibody-enriched B or T cells, or other cell types. Cells have an intact plasma membrane, so they do not leak excessive amounts of mRNA into the surrounding medium. Cells need not be alive.

[0344] Cells were washed twice by centrifugation (200 g, 10 min for T cells or B cells) in cell buffer 1 x Dulbecco's phosphate buffered saline (PBS). Cells were then diluted to 3.5x10 in cell buffer 6 / mL cell concentration. The suspension was then pipetted through a 20 μm cell strainer.

Embodiment 1b

[0345] Example 1b - Protocol for preparation of solid tissues for emulsion-based large-scale high-throughput single-cell polynucleotide sequencing.

[0346] Solid tissues (e.g., tumor or non-tumor biopsies) were treated with various proteases including collagenase III (200 U / mL), DNase I (200 U / mL), and trypsin (5 mg / mL) and NEDB (Invitrogen) ) to produce mixtures of individual cells and aggregates containing more than one cell. Briefly, tumors removed from mice were added to cold medium, and the surrounding mouse mammary gland tissue and fat were removed. Tumors were cut into 2-4 mm pieces, which were then incubated with appropriate dissociation solutions or enzymes for 30 min at 37°C. Tumor fragments were then mixed up and down every 10 min using a 1,000 mL micropipette tip cut to the appropriate diameter for the tissue fragment size. After each incubation period, debris was filtered through a 40 mm nylon mesh cell strainer. The released cells were centrifuged at 1200 r...

Embodiment 2

[0348] Example 2 -For large-scale, high-throughput, single-cell polynucleotide sequencing based on emulsions

[0349] Scheme for the preparation of the emulsion reaction mixture.

[0350] Mix the emulsion reaction mixture containing the reagents and oligonucleotides in Table 1 below in a PCR-clean cabinet at room temperature.

[0351] Table 1

[0352]

[0353]

[0354] / 5Biosg / = 5' biotin modification; / iSp18 / = 18-carbon spacer; V = A, C or G; N = any base; rG = riboguanosine; W = A or T.

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Abstract

Provided herein are methods and composition for immune repertoire sequencing and single cell barcoding. The methods and compositions can be used to pair any two sequences originating from a single cell, such as heavy and light chain antibody sequences, alpha and beta chain T-cell receptor sequences, or gamma and delta chain T-cell receptor sequences, for antibody and T-cell receptor discovery, disease and immune diagnostics, and low error sequencing.

Description

[0001] cross reference [0002] This application claims priority to US Provisional Application No. 62 / 050,549, filed September 15, 2014, and US Provisional Application No. 62 / 051,832, filed September 17, 2014, each of which is incorporated herein by reference in its entirety. Background technique [0003] Current antibody display technologies (phage, yeast, ribosomal, mammalian, etc.) are limited because the quality of the selected antibody candidates is limited by the starting library from which they were generated. Approaches such as combinatorial and "smart" antibody design approaches and hybridoma discovery approaches often result in synthetic antibodies that exhibit downstream complications including difficulties in large-scale expression, high risk of immunogenicity in patients, and in addition to high binding affinity lack of adequate immune function. A very small number of antibodies derived from the display technology have successfully passed clinical trials in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1093C12Q1/6874C12Q2525/179C12Q2563/179C12Q2565/514C12Q2535/122C12Q2563/185C12Q1/6869C12Q2563/159
Inventor 弗朗索瓦·维格纳尔特阿德里安·阮格汉姆布里格斯克里斯多佛·赖安·克劳泽斯蒂芬·雅各布·戈德弗莱斯索尼亚·汀布莱克
Owner ABVITRO LLC
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