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Deep sequencing profiling of tumors

A technology for sequencing and tumor, applied in the field of deep sequencing profiling analysis of tumors, which can solve problems such as elevation

Inactive Publication Date: 2018-08-28
VENTANA MEDICAL SYST INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The risk of introducing bias to the raw information is considered to increase when using a smaller number of molecular individuals as input in the amplification process and when the amount of amplification is increased (i.e., a greater number of PCR cycles is applied)

Method used

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  • Deep sequencing profiling of tumors
  • Deep sequencing profiling of tumors
  • Deep sequencing profiling of tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 - Sample library preparation using KAPA HyperPlus library preparation

[0127] 1.1. Resuspend lyophilized indexing adapters (Adapter Kit A).

[0128] 1.1.1. Spin down the lyophilized indexing adapters contained in SeqCap Adapter Kit A and / or B briefly to allow the contents to pellet at the bottom of the tube.

[0129] 1.1.2. Add 50 microliters of cold PCR grade water to each of the 12 tubes labeled 'SeqCap Index Adapter' in SeqCap Adapter Kit A and / or B. Keep adapters on ice.

[0130] 1.1.3. Briefly vortex the index adapters with PCR grade water and swirl down to resuspend the index adapter tubes.

[0131] 1.1.4. Tubes of resuspended index adapters should be stored at -15 to -25°C.

[0132] 1.2. Preparation of sample library

[0133] 1.2.1. Dilute the gDNA to be used for library construction (about 100 ng to about 1 microgram) in 10 mM Tris-HCl (pH 8.0) to a total volume of 35 microliters in a 0.2 ml tube or well of a PCR plate.

[0134] 1.2.2. Assemble...

Embodiment 2

[0178] Example 2 - Hybridization Sample and SeqCap EZ Probe Pool

[0179] 2.1.1. Allow AMPure XP Reagent to warm to room temperature for at least 30 minutes before use.

[0180] 2.1.2. Add 5 microliters of COT Human DNA (1 mg / ml) contained in the SeqCap EZ Accessory Kit v2 to a new tube / well.

[0181] 2.1.3. Add 3 μg DNA sample library to sample containing 5 μl COT human DNA. Multiple libraries constructed from the same sample can be combined for this purpose.

[0182] 2.1.4. Add 2,000 pmol (or 2 µl) of Hybridization Enhancer Oligomer (1 µl of 1,000 pmol SeqCap HE Universal Oligomer and 1 µl of 1,000 pmol SeqCap HE Indexing Oligomer matching the sample library adapter index) Add to DNA sample library plus COT human DNA.

[0183] 2.1.5. Determine the total volume of the above mixture by adding the input volumes of COT Human DNA, DNA Sample Library Pool, SeqCap HE Universal Oligomer and SeqCap HE Index Oligomer Pool.

[0184] 2.1.6. Add 2 volumes of AMPure XP Reagent (equi...

Embodiment 3

[0204] Example 3 - Cleaning and Recovery of Captured DNA Sample Libraries

[0205] 3.1. Dilute the 10X Wash Buffer (I, II, III and Stringent) and 2.5X Bead Wash Buffer contained in the SeqCap Hybridization and Wash Kit to create a 1X working solution. The volumes listed below are sufficient for one subcapture.

[0206]

[0207] 3.2. Preparation of capture beads

[0208] 3.2.1. Allow the capture beads contained in the SeqCap Pure Capture Bead Kit to equilibrate to room temperature for 30 minutes before use.

[0209] 3.2.2. Vortex the capture beads for 15 seconds prior to use to ensure a homogeneous mixture of beads.

[0210] 3.2.3. Aliquot 50 microliters of beads for each capture into 0.2ml or 1.5ml tubes (ie use 50 microliters of beads for one capture and 200 microliters of beads for four captures, etc.). Sufficient beads for two captures and twelve captures can be prepared in one 0.2 ml tube and 1.5 ml tube, respectively.

[0211]3.2.4. Place the tube on the magnetic...

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Abstract

In one aspect of the present invention is a targeted sequencing workflow, wherein an input sample comprising a sufficient quantity of a genomic material is provided such that minimal or no amplification cycles are utilized prior to sequencing.

Description

[0001] Cross references to related applications [0002] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 415,952, filed November 1, 2016, and U.S. Provisional Patent Application No. 62 / 279,126, filed January 15, 2016, according to The entire text of its publication is incorporated herein by reference. field of invention [0003] The present disclosure provides a targeted representational sequencing workflow. Background of the invention [0004] Current diagnostic oncology utilizes information obtained from a subset of tumors and is predicted on the assumption that tumors are composed of cells that are uniform in their composition. Rather than being uniform in composition, many tumors are heterogeneous. Indeed, some solid tumors have been reported that are not homogeneous but consist of multiple genetically distinct, spatially segregated populations of cancer cells. See Gerlinger et al., NEJM (2012) 366:883-92; and Yachi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q1/6806C12Q2527/146C12Q2531/113C12Q1/6874
Inventor N·亚历山大D·布格斯S·斯塔尼斯劳H·罗森鲍姆
Owner VENTANA MEDICAL SYST INC
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