DNA barcode compositions and methods of in situ identification in a microfluidic device

A barcode, object technology, applied in the fields of biochemical equipment and methods, DNA preparation, chemical instruments and methods, etc., can solve the problems of difficulty and impossibility of specific phenotype association

Active Publication Date: 2019-08-09
BERKELEY LIGHTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite these advances, linking genome and transcriptome sequences to the specific phenotypes of the cells being sequenced remains difficult—and often impossible

Method used

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  • DNA barcode compositions and methods of in situ identification in a microfluidic device
  • DNA barcode compositions and methods of in situ identification in a microfluidic device
  • DNA barcode compositions and methods of in situ identification in a microfluidic device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0467] Example 1. RNA capture, sequencing library preparation and sequencing results as demonstrated in OKT3 cells.

[0468] Cells: OKT3 cells, a murine myeloma hybridoma cell line, were obtained from ATCC ( Catalog Number CRL-8001 TM ). Cells are provided as suspension cell lines. By inoculating about 1 x 10 5 to about 2×10 5 viable cells / mL and maintain the culture by incubating at 37°C with 5% carbon dioxide in air as the atmosphere. Flask cells every 2-3 days. Count the OKT3 cell number and viability, and adjust the cell density to 5 × 10 5 / ml to load into the microfluidic device.

[0469] Culture medium: 1000ml Iskoff's modified medium ( Cat. No. 30-2005), 200ml fetal bovine serum ( Cat. No. 30-2020) and 10ml penicillin-streptomycin ( Cat. No. 15140-122) were combined to prepare media. Filter the complete medium through a 0.22 μm filter and store at 4 °C in the dark until use.

[0470] When perfusing during incubation, the medium was continuously conditio...

Embodiment 2

[0481] Example 2. T cell phenotyping, culture, measurement and RNA sequencing. Association of phenotypes with genomic information.

[0482] The microfluidic system, materials, and methods were the same as in Experiment 1, with the following differences:

[0483] Cells: Control cells were human peripheral blood T cells. Sample cells are human T cells derived from a human tumor sample.

[0484] Medium: RPMI 1640 medium (Gibco, #12633-012), 10% fetal bovine serum (FBS), (Seradigm, #1500-500); 2% human AB serum (Zen-bio, #HSER-ABP100ml), IL-2 (R&D Systems, 202-IL-010) 2U / ml; IL-7 (PeproTech, #200-07) 10ng / ml; IL-15 {PeproTech, #200-15) 10ng / ml, IX Pluronic F -127 (Life Tech Cat. No. 50-310-494).

[0485] Human T cells derived from human tumor samples were stained off-chip with antigen and then introduced into the microfluidic channels of the OptoSelect device at a density of 5×E6 cells / ml. Antigen-positive T cells (P-Ag) and antigen-negative cells (N-Ag) were moved by light-d...

Embodiment 3

[0493] Example 3. DNA capture, sequencing library preparation and sequencing results as demonstrated in OKT3 cells. Unless otherwise noted in this example, equipment, priming and perfusion protocols, cell sources and preparation were used / performed as in the general methods above. Media and OptoSelect devices were maintained at 37°C unless otherwise stated.

[0494] Table 4. Primers used in this experiment.

[0495]

[0496]

[0497] This experiment demonstrates that Nextera sequencing libraries (Illumina) can be generated using isothermal PCR with a biotinylated priming sequence (carrying the barcode) attached to a bead and a primer free in solution. OKT3 cells (150) were input into the OptoSelect device and loaded into the NanoPen chamber using light-driven dielectrophoretic force. Figure 24 Cells are shown after delivery to the NanoPen chamber. Light-driven dielectrophoretic force delivers one cell to each of the seven NanoPen chambers, where misses deliver one cell...

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Abstract

Apparatuses, compositions and processes for DNA barcode de-convolution are described herein. A DNA barcode may be used to provide a bead specific identifier, which may be detected in situ using hybridization strategies. The DNA barcode provides identification by sequencing analysis. The dual mode of detection may be used in a wide variety of applications to link positional information with assay information including but not limited to genetic analysis. Methods are described for generation of bar coded single cell sequencing libraries. Isolation of nucleic acids from a single cell within a micro fluidic environment can provide the foundation for cell specific sequencing library preparation.

Description

[0001] This application is a nonprovisional application, U.S. Provisional Application No. 62 / 403,116 filed October 1, 2016; U.S. Provisional Application No. 62 / 403,111 filed October 1, 2016, pursuant to 35 U.S.C. 119(e); U.S. Provisional Application No. 62 / 457,399, filed February 10, 2017; U.S. Provisional Application No. 62 / 457,582, filed February 10, 2017; Priority of U.S. Provisional Application No. 62 / 470,669, filed March 13, 2017 , each disclosure is hereby incorporated by reference in its entirety. Background technique [0002] The advent of single-cell genome amplification techniques and next-generation sequencing approaches has led to breakthroughs in our ability to sequence the genome and transcriptome of individual biological cells. Despite these advances, linking genome and transcriptome sequences to the specific phenotypes of the cells being sequenced remains difficult—and often impossible. As further described herein, the ability to decipher barcodes, such as DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B20/04C40B40/06C40B70/00B01J19/00
CPCC40B20/04C40B40/06C40B70/00B01J2219/00547B01J2219/00572B01J2219/00576C12Q1/6806C12Q1/6841C12Q2525/185C12Q2563/179C12Q2565/629C12Q2535/122G16B25/00G16B30/00C07K14/705C12N15/1093C12N15/1096C12Q1/6813C12Q2565/125C12Q2565/519C12Q2565/537C12Q2563/107C12Q2525/131B01J19/0046B01L3/502707B01L2300/0877B01L2400/0424C12N15/1065C12Q1/6809
Inventor M·苏米隆H·M·贝内特Y·X·梅西亚冈萨雷斯M·S·托赫R·K·拉梅纳尼
Owner BERKELEY LIGHTS INC
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