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DNA library construction kit based on illumina sequencing platform, library construction method and application

A DNA library and library construction technology, applied in the field of DNA library construction kits, can solve the problems of low amplification efficiency, high price of finished kits, low success rate of library construction, etc.

Pending Publication Date: 2020-04-14
江西海普洛斯医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the above-mentioned library construction kits have the following defects in library construction: (1) The cost of library construction is high: the price of finished kits is high, resulting in a substantial increase in sequencing costs; (2) The experimental process is relatively time-consuming: for Fragmented DNA is filled in at the ends, and A is added to both ends of the filled-in fragments. This process is performed separately and takes a long time, such as KAPAHyper Prep Kit It takes about 1 hour for the platform to perform this step; (3) The process is cumbersome: the adapter added by the NEB kit is a U-shaped adapter. After adding the adapter, it is necessary to use USER enzyme to change the U-shaped adapter into a Y-shaped adapter. The experimental process is relatively cumbersome; ( 4) Low amplification efficiency: For samples with low sample size, the success rate of library construction is low
[0004] Therefore, the existing kits are still difficult to meet the diverse needs of DNA library construction in the market, and it is necessary to provide a new library construction kit with low cost, short time consumption, simple process and high success rate

Method used

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  • DNA library construction kit based on illumina sequencing platform, library construction method and application
  • DNA library construction kit based on illumina sequencing platform, library construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Genomic DNA Fragmentation

[0078] In this example, after using Qubit3.0 to quantify genomic DNA, add 300ng of sample into a 0.2mL PCR tube, add 0.1×TE buffer to make up to 55μL, mark the sample number on the tube cap, mix well and centrifuge; open Ultrasonic cleaner, set the ultrasonic time of the ultrasonic cleaner to 5min, and place 0.2mL PCR tubes containing samples in order around the ultrasonic sounding position, and interrupt 9 samples at a time; after ensuring that the PCR tube is clamped, inject pure water to the ultrasonic wave. At the 2 / 3 scale of the cleaning instrument, click the start button to perform ultrasound interruption. The parameter settings are shown in Table 1. After the ultrasound is completed, the fragmented DNA is purified with AMpure XP magnetic beads and dissolved in 39.5 μL NF water.

[0079] Table 1 Ultrasonic treatment parameters

[0080] Expected fragment length (bp) Power (W) temperature(℃) Ultrasonic time (s) ...

Embodiment 2

[0081] Example 2 End Repair and Add A Reaction

[0082] Prepare the end-repair reaction solution according to Table 2 to complete the end-repair and A addition reaction of fragmented DNA; blow and mix with a pipette, collect the reaction solution to the bottom of the tube after brief centrifugation, and perform end-repair and A addition according to the conditions in Table 3. A response.

[0083] Table 2 End repair and A reaction system

[0084] Reagent Volume (μL) Fragmented DNA 37 end repair buffer 10 end repair enzyme 3

[0085] Table 3 End Repair and Add A Reaction Conditions

[0086] temperature Reaction time 22℃ 10min 72℃ 10min 4℃ ∞

Embodiment 3

[0087] Embodiment 3 linker ligation reaction

[0088] Prepare the adapter ligation reaction solution according to Table 4 to complete the adapter ligation of the fragmented DNA, wherein the adapter set working solution is self-synthesized P5 (SEQ ID NO: 6-10) and P7 adapter sequence (SEQ ID NO: 6-10) with a molar ratio of 1:1. ID NO: 1-5), the mixed sequence diluted to 10 μM; use a pipette to mix well, centrifuge briefly to collect the reaction solution to the bottom of the tube, and perform the adapter ligation reaction according to Table 5.

[0089] Table 4 Adapter Ligation Reaction System

[0090] Reagent Volume (μL) end repair plus A product 50 ligation buffer 10 T4 DNA ligase 2 Linker set working solution (10μM) 3

[0091] Table 5 Adapter Ligation Reaction Conditions

[0092] temperature Reaction time 22℃ 15min 4℃ ∞

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Abstract

The invention provides a DNA library construction kit based on an illumina sequencing platform, a library construction method and application. The kit comprises a P7 linker, a P5 linker, an enzyme composition or a buffer solution, wherein the enzyme composition comprises any one or a combination of at least two of a terminal repair enzyme, a PCR amplification enzyme or a T4DNA ligase; and the buffer solution comprises a tail end repairing buffer solution and / or a connecting buffer solution. According to the kit disclosed by the invention, a reaction solution is prepared by adopting commercially available reagents, so that the library construction cost is reduced, the reaction procedure is simplified, a terminal repair reaction system and an A-adding reaction system are combined in one system, the reaction time is shortened, and the enzyme connection efficiency is improved.

Description

technical field [0001] The invention belongs to the technical field of sequencing, and relates to a DNA library construction kit based on an illumina sequencing platform, a library construction method and an application. Background technique [0002] Currently, the library construction kits suitable for the Illumina NGS platform on the market mainly include Illumina original kits, KAPA Hyper Prep Kit platforms, IDT NanoPrep DNA Library Construction Kit and NEBNext Ultra II DNA Kit, etc. The main process of library construction using the above kit is as follows: fragment the genome; fill in the ends of the fragmented DNA; add A to both ends of the filled-in fragments; add adapters to both ends of the fragments by TA ligation (NEBNext Ultra II DNA Kit is to add U-shaped adapters at both ends of the fragments, and use USER enzyme to open the U-shaped adapters); use AMPure XP magnetic beads to capture the fragments, and obtain the target fragments with adapters added; after PC...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6806
CPCC40B50/06C12Q1/6806C12Q2531/113C12Q2525/191C12Q2523/301C12Q2521/501
Inventor 刘江辉马焕班卢超吕艳花王伟凤
Owner 江西海普洛斯医学检验实验室有限公司
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