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Primer suitable for sequencing DNA-coding compound sequencing library

A technology for sequencing primers and compound libraries, which is applied in the field of primers for DNA-encoded compound sequencing libraries to construct sequencing, can solve the problems of low sequencing quality, influence authenticity, poor base diversity, etc., to improve sequencing quality, reduce detection costs, and improve The effect of data utilization

Pending Publication Date: 2020-04-07
HITGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The base diversity of conventional genomic DNA is good, while the base diversity used in DNA-encoded compound libraries is poor, and the sequencing quality of DNA-encoded compounds is low when using conventional sequencing methods
In addition, the conventional library construction method first amplifies the double-stranded DNA of the DNA-encoded compound, then adds A to the end repair, connects the sequencing adapter and PCR amplification to form a sequencing library. This method requires four steps, especially multi-step PCR will introduce more Amplification bias, which affects the authenticity of DNA-encoded compounds in screening samples enriched for compounds corresponding to DNA molecules

Method used

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  • Primer suitable for sequencing DNA-coding compound sequencing library
  • Primer suitable for sequencing DNA-coding compound sequencing library
  • Primer suitable for sequencing DNA-coding compound sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Establishment of a DNA-encoded compound sequencing library by conventional methods

[0038] 1. PCR amplification

[0039] According to the following amplification primer sequences and amplification methods, the screened DNA-encoded compounds were amplified, and two biological replicates were set up, labeled S1 and S2 respectively, and a negative control was set at the same time.

[0040]

[0041]

[0042] The PCR amplification system is 30 μL of screened DNA-encoded compounds, 4 μL of 5’-primers, 4 μL of 3’-primers, 100 μL of Q5 hot start ultra-fidelity 2X master mix and 62 μL of ddH 2 O, a total of 200 μL per sample.

[0043] The PCR reaction program is: first step: 98°C for 10 minutes; second step: 98°C for 10 seconds, 55°C for 10 seconds, 72°C for 10 seconds, and repeat 18 times; third step: 72°C for 10 seconds Cool to 4 °C after 1 min.

[0044] Sampling and electrophoresis to detect PCR products. Take 5 μL of each sample for agarose gel electroph...

Embodiment 2

[0056] Example 2 Establishment of a DNA-encoded compound sequencing library by the method of the present invention

[0057] According to the following amplification primer sequences and amplification methods, amplify the same screened DNA-encoded compound as in Example 1, set up two biological repeats, respectively marked as S1-ONE and S2-ONE, and set negative control.

[0058] 1. Primers:

[0059]

[0060] 2. How to build a database:

[0061] The PCR amplification system was 30 μL of screened DNA-encoded compounds, 4 μL of 5’-primers, 4 μL of 3’-primers, 100 μL of Q5 hot-start ultra-fidelity 2X master mix and 62 μL of ddHO, a total of 200 μL for each sample.

[0062] The PCR reaction program is: first step: maintain 98°C for 10 minutes; second step: maintain 98°C for 30 seconds, 70°C for 30 seconds, 72°C for 30 seconds, and repeat 18 times; third step: maintain 72°C for 10 seconds Cool to 4 °C after 1 min.

[0063] Take 5 μL of each sample for agarose gel electrophores...

Embodiment 3

[0070] Example 3 Comparison of the sequencing quality between the conventional library construction method without linker sequence and the library construction method of the present invention with linker sequence

[0071] In next-generation sequencing, a corresponding quality value is given for each base measured, and this quality value is used to measure the accuracy of sequencing. The higher the quality value (Q), the smaller the probability (P) that the base is detected incorrectly, and its calculation formula is Q=-101gP. Therefore, when the Q≥30 of a single base (sometimes also written as "quality ≥ Q30"), it means that the probability of the base being detected incorrectly is less than 1‰. For the entire sequencing reads (reads), the sequencing quality is generally considered to be good when the proportion of bases with sequencing quality > Q30 is greater than 85%.

[0072] The following will compare the Q30 ratio obtained by the corresponding sequencing reaction of the...

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Abstract

The invention discloses a sequencing primer suitable for sequencing a DNA coding compound library. The sequencing primer is a sequence formed by connecting a DNA coding compound amplification primer sequence, a connector sequence and a sequencing primer sequence. The primer can improve the sequencing quality of the DNA coding compound library, one-step sequencing can further be realized, and the application prospects are excellent.

Description

technical field [0001] The invention discloses a primer suitable for the construction and sequencing of DNA coded compound sequencing library. Background technique [0002] DNA-encoded compound library technology combines combinatorial chemistry and molecular biology techniques. By linking a fragment compound with a unique sequence of DNA at the molecular level, the "combination-disassembly" strategy of combinatorial chemistry can be quickly passed through two or more cycles. to construct a large number of compound libraries. Wherein, each compound in the compound library is composed of different fragment compounds, and is coded and identified by the DNA of the corresponding specific base sequence. At present, the scale of DNA-encoded compound libraries used in industry can reach hundreds of billions to trillions, and can be identified by screening and sequencing methods. This technology makes the screening of lead compounds faster and more efficient than ever, and has bec...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12N15/11C40B50/06C40B40/06
CPCC12Q1/6869C40B50/06C40B40/06C12Q2531/113C12Q2535/122
Inventor 李进刘建刘承忠王枫陈秋霞李游窦登峰
Owner HITGEN INC
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