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Next generation sequencing library building technology based on multiplex PCR (Polymerase Chain Reaction)

A technology for a second-generation sequencing library and a construction method, which is applied in the field of second-generation sequencing library construction, can solve the problems of increased time cost, high economic cost, and reduction, and can reduce the difference in amplification efficiency, reduce the type and quantity of primers, inhibit the The effect of mutual interference

Inactive Publication Date: 2017-03-15
上海美迪维康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest defect of this product is that the production cycle of customized panel design is long, which takes two to three months, which seriously increases the time cost; and the cost of building a database for each sample is as high as 1000-2000 yuan, which makes it The economic cost is extremely high; in addition, the kit is only applicable to the Ion Torrent sequencing platform, which is greatly affected by platform factors and is not conducive to the reduction of sequencing costs

Method used

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  • Next generation sequencing library building technology based on multiplex PCR (Polymerase Chain Reaction)
  • Next generation sequencing library building technology based on multiplex PCR (Polymerase Chain Reaction)
  • Next generation sequencing library building technology based on multiplex PCR (Polymerase Chain Reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Fragmentation of Genomic DNA

[0027] a. Turn on the Qsonica ultrasonic breaker, let the circulating water bath cool down in advance, and the working temperature is 4°C.

[0028] b. Transfer a total of 1ug of genomic DNA to a 0.2mL centrifuge tube, make up to 100uL with Nuclease Free Water (hereinafter referred to as NFW), vortex to mix, and centrifuge for a short time.

[0029] c. Put a 0.2mL centrifuge tube into the adapter of the sonicator, tighten the central shaft, insert the top cover, close the machine, and set the interruption program: amplitude -25%, on & off 20s-30 seconds, duration 15 minutes. Start interrupting.

[0030] d. After the interruption, take out the 0.2mL centrifuge tubes, arrange them neatly in order, and open the caps.

Embodiment 2

[0031] Example 2. 3in 1 connector connection

[0032] a. Configure the reaction system 22uL, first configure the reagents other than DNA, according to 125% of the theoretical value, shake and mix, centrifuge for a short time, dispense into a 96-well plate, add 7uL to each well, and then add 0.2mL from the front Transfer 15uL of DNA to each well in a centrifuge tube and cover with a cap. Vortex to mix and centrifuge briefly.

[0033]

[0034] b. Put the 96-well plate or eight tubes into the PCR machine. Setting program: 30 minutes at 25°C, 15 minutes at 72°C, hold at 4°C.

[0035] c. After finishing, add 0.5uL T4 Ligase (single gun) and 1uL Adapter to each well, and cover with a new cap. Vortex to mix and centrifuge briefly. Incubate at room temperature for 20-30 minutes.

[0036] d. Shake the magnetic beads for 30 seconds. After mixing thoroughly, add 25uL magnetic beads to each well and cover with a new cap. Vortex to mix and centrifuge briefly. Incubate for 5 minutes...

Embodiment 3

[0041] Example 3. Multiplex PCR

[0042] 1. The first round of multiplex PCR

[0043] a. Check the sample concentration according to the Qubit dsDNA HS Assay Kit operation manual. According to the sample concentration detected by Qubit, take the same amount (10ng) for each sample, mix every 5-10 samples and transfer them to a new well plate.

[0044] b. The first round of specific primers are ordinary PCR amplification primers, with a total of about 20 bases, the Tm value is set at 60°C, and the sequence is designed according to the target region. Multiple primers are mixed into an OP primer pool as the first Upstream primers for one round of PCR. Configure the total PCR system according to 125% of the theoretical value, shake and mix, and centrifuge for a short time.

[0045]

[0046] c. Put the orifice plate into the PCR instrument, and select the MAPlex-1st program to run for 2 hours.

[0047] 2. Purification of PCR products

[0048] a. After PCR, remove the well pla...

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Abstract

The invention relates to the technical field of biology and particularly relates to a next generation sequencing library building technology. A library is built through utilizing two rounds of multiplex PCR (Polymerase Chain Reaction). Aiming at each target region or target site, two specific primers are designed; and an upstream primer OP is used for the first round of multiplex PCR and a downstream primer IP is used for second round of multiplex PCR. After a product of the second round of PCR is purified, Q-PCR quantification is carried out; and then the product is diluted to a proper concentration and is used for next generation sequencing. According to the next generation sequencing library building technology, the problems of an Ampliseq kit can be effectively reduced, and a customization-oriented panel design production period only needs two weeks; the cost of building a library by a single sample is reduced; and the technology is general for Ion Torrent and Illumina sequencing platforms.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a technology for constructing a next-generation sequencing library. Background technique [0002] Due to its ultra-high sequencing capability, next-generation sequencing has extremely important applications in scientific research and clinical practice. Next-generation sequencing technology is also called deep sequencing and massively parallel sequencing. The core idea is Sequencing by Synthesis (Sequencing by Synthesis), that is, to determine the DNA sequence by capturing the markers of the newly synthesized ends. The existing technology platforms mainly include Roche / 454FLX, Illumina / Solexa Genome Analyzer, and Applied Biosystems SOLIDsystem. These three technology platforms have their own advantages. The sequencing fragments of 454FLX are relatively long, and the high-quality read length can reach 400bp; Solexa sequencing is the most cost-effective, not only the price of the mach...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/68
CPCC40B50/06C12N15/1093C12Q1/6869C12Q2525/191C12Q2537/143C12Q2535/122
Inventor 王筱恬徐峰
Owner 上海美迪维康生物科技有限公司
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