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A linker sequence and its application

A joint sequence and sequencing joint technology, which is applied in the field of molecular biology, can solve problems such as low accuracy, increase experimental complexity and cost, achieve low data bias rate, improve sequencing accuracy, and avoid point mutation errors

Active Publication Date: 2021-04-13
CAPITALBIO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used methods for removing repetitive sequences include: aligning the reads to positions in the reference genome, such as the start site, to remove repetitive reads. The advantage of this method is that it does not increase the complexity and cost of the experiment. The disadvantage is that the accuracy is low. Because there are situations where the reads aligned to the same position in the reference genome do not necessarily originate from the same DNA

Method used

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  • A linker sequence and its application
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  • A linker sequence and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] (1) Genomic DNA extraction

[0068] The MagPure Genomic DNA Extraction Kit was used to extract DNA from 20 samples, and the extracted DNA was stored in a -20°C refrigerator for a short period of time and stored in a -80°C refrigerator for a long time;

[0069] (2) Fragmentation and end repair

[0070] The extracted DNA sample was fragmented and end-repaired by one-step reaction using WGS-IT Frag enzyme from Qiagen Company, the reagents were mixed and centrifuged, placed on an ice box, and the reaction system was prepared according to Table 1;

[0071] After mixing, put it in a PCR machine for reaction, the reaction conditions are: 4°C for 1min, 32°C for 15min, 65°C for 30min, and store at 4°C;

[0072] After the reaction is completed, the reaction product is centrifuged briefly for the next step of ligation reaction.

[0073] Table 1

[0074] Reagent Dosage dna sample 500ng 10×WGS IT Buffer 5μL 5×WGS IT Frag 10μL pure water Mak...

Embodiment 2

[0083] Compared with Example 1, this example also includes the step of performing PCR amplification on the library. The PCR amplification system is shown in Table 3, and the conditions are: 72°C for 5 minutes, 98°C for 2 minutes, 98°C for 20s, 58°C for 30s, 72°C for 30s, 4 cycles, 72°C for 5min, 16°C storage;

[0084] After the reaction, use the magnetic bead method to purify the product, keep the supernatant at 0.8×, keep the magnetic beads for 1.2× magnetic bead purification, dissolve in 20 μL, and then detect the library concentration;

[0085] table 3

[0086] Reagent Dosage library 20 μL 2x HiFi PCR Master Mix 25 μL Primer pair 5μL

Embodiment 3

[0088] Compared with Example 2, the molecular tag part is not included in the linker sequence, and other conditions are the same as Example 2.

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Abstract

The present invention provides an adapter sequence and its application. The adapter sequence includes a molecular tag composed of several bases and a sequencing adapter based on a sequencing platform; the molecular tag is located at the 3' end of the sequencing adapter; the adapter sequence Attached to fragmented DNA via molecular tags. The present invention combines molecular tags containing several bases with sequencing adapters based on different sequencing platforms, and the constructed adapter sequences are connected to one end and / or both ends of the fragmented DNA in the early stage of library construction to realize the identification of the original DNA fragments. Marking effect, DNA is directly sequenced on the machine after the sequencing adapter is connected, the original sequence is retained, and the technical effect of tracing the original source of the DNA fragment is realized. During the analysis of the sequencing result, the repeated fragment in the read length is removed according to the molecular label, which improves the Sequencing accuracy.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a linker sequence and its application, in particular to a linker sequence and its application in the construction of a library and the preparation of a screening kit for carriers of monogenic genetic diseases. Background technique [0002] Carrier screening refers to the use of economical and accurate methods to screen out carriers with normal phenotypes from the population when the incidence of a certain genetic disease is high in a specific population, in order to prevent the further development of the disease in the population , to carry out risk assessment and guidance on marriage and childbearing. The term "carrier" has a broad meaning in the medical field. In the field of genetic diseases, it mainly refers to individuals who carry a disease-causing gene (heterozygous state), but are still healthy until the time of testing. [0003] Carrier screening can provide ris...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6883C12N15/10C40B50/06C40B70/00C40B80/00
CPCC12N15/1093C12Q1/6806C12Q1/6883C12Q2600/156C40B50/06C40B70/00C40B80/00C12Q2525/191C12Q2565/519
Inventor 黄铨飞景丽芳王杨刘情黄晓燕
Owner CAPITALBIO GENOMICS
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