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Co-library-building method capable of distinguishing DNA and RNA sources

A source and library technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of inability to distinguish whether the sequencing results come from DNA or RNA, the inability to distinguish the specific source of sequencing information, and increasing the number of unknown pathogens The difficulty of detection and other issues, to achieve the effect of reducing gene fusion and gene mutation events, improving connection efficiency, and reducing self-connection rate

Pending Publication Date: 2021-09-07
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method cannot distinguish whether the sequencing results are derived from DNA or RNA, so it has great application limitations
Especially for the field of tumor detection, the low fidelity of reverse transcriptase will introduce additional gene mutations, which brings a lot of false positive interference to the identification of tumor process. The current DNA and RNA co-construction library sequencing technology cannot be applied to tumor diagnostically
Because the current DNA and RNA co-construction method cannot distinguish the specific source of sequencing information, in the field of identification of unknown pathogens, only genetic evolution analysis of bioinformatics and further experimental verification can be used to determine whether the unknown pathogen is a DNA pathogen or an RNA pathogen. This increases the difficulty of detecting unknown pathogens

Method used

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  • Co-library-building method capable of distinguishing DNA and RNA sources
  • Co-library-building method capable of distinguishing DNA and RNA sources
  • Co-library-building method capable of distinguishing DNA and RNA sources

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Establishment of DDRC-seq process

[0066] In this example, we set up the process of DDRC-seq, the flow chart is shown in figure 1 and figure 2 . The specific method is as follows:

[0067] 1) DNA and RNA fragmentation:

[0068] Table 2

[0069] components Dosage DNA & RNA 100ng fragmentation buffer 4μL DSN (Evrogen) 2μL Total 17μL

[0070] Fragmentation buffer: 200mM Tris-HCl, 300mM KCl, 20mM MgCl 2 , pH 8.3.

[0071] React at 37°C for 20 minutes, react at 85°C for 15 minutes, and store at 4°C.

[0072] 2) RNA end repair, polyadenylation

[0073] table 3

[0074] components Dosage The above reaction system 17μL T4 PNK (Yeasen, 12902) 1μL E.coli Poly(A) Polymerase(NEB,M0276S) 2μL Total 20 μL

[0075] React at 37°C for 30 minutes.

[0076] 3) rRNA removal, reverse transcription and template switching

[0077] Table 4

[0078] components Dosage ...

Embodiment 2

[0104] Example 2: Effect of DDRC-seq on library construction with different DNA inputs

[0105] In this example, we verified the library construction effect of DDRC-seq under the condition of calf thymus 0.1ng-100ng gDNA input. The experimental procedure is shown in Example 1. The schematic diagram is shown in image 3 , see Table 9 for the number of PCR cycles and library yield, and see Table 9 for the distribution of library sizes. Figure 4 , for the analysis of sequencing results, see Figure 5-7 .

[0106] Table 9

[0107]

[0108] The results are shown in Table 9 and Figure 4-5 As shown, DDRC-seq has good library yield and library size for 0.01-100ng calf gDNA input. like Image 6 and Figure 7 , in the DNA sequencing data, the calf genome alignment rate of the library sequencing data of 100ng DNA input amount reaches 99.64%, among which there is a co-constructed library RNA structure (G 7-11 NN…NNA 18-25 ) data accounted for only 0.007%; the comparison rate ...

Embodiment 3

[0109] Example 3: Effect of DDRC-seq on library construction with different RNA inputs

[0110] In this example, we verified the library construction effect of DDRC-seq under the condition of HEK293F 0.1ng-1000ng RNA input. The experimental procedure is the same as that in Example 1. The schematic diagram is shown in Figure 9 , see Table 10 for the number of PCR cycles and library yield, see Table 10 for library size distribution Figure 10 , for the analysis of sequencing results, see Figure 11-16 .

[0111] Table 10

[0112]

[0113] The results are shown in Table 10 and Figure 10-11 As shown, DDRC-seq has good library yield and library size for 0.1-1000ng human RNA input. like Figure 11-Figure 15 , in the DNA sequencing data, the human transcriptome alignment rate of the library sequencing data of 1000ng RNA input amount reaches 99.34%, among which there is a co-constructed library RNA structure (G 7-11 NN…NNA 18-25 ) data accounted for 99.32%, compared to the...

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Abstract

The invention provides a co-library-building method capable of distinguishing DNA and RNA sources. The co-library-building method is characterized by comprising the following steps of: in a library building process, firstly fragmenting DNA and RNA, adding polyA at the tail end of RNA, in a reverse transcription process, adding Poly (dC) at the other tail end of transcribed cDNA, respectively adding p5 and p7 connectors at two ends of cDNA, then adding p5 and p7 connectors at two ends of DNA, and carrying out library amplification and sequencing on DNA and cDNA. The co-library-building method provided by the invention has the advantages that two ends of a cDNA fragment of an RNA source can carry a section of fixed nucleotide sequence for distinguishing the source of sequencing data, so that independent library building and sequencing do not need to be carried out on DNA and RNA in a sample, and cost of NGS detection can be effectively reduced.

Description

technical field [0001] The patent of the present invention relates to a co-construction library method that can distinguish the source of DNA and RNA, which belongs to the field of biotechnology. Background technique [0002] High-throughput sequencing technology (High-throughput sequencing), also known as "Next-generation" sequencing technology (NGS), is a large-scale parallel sequencing technology that can simultaneously perform hundreds of thousands to several million Sequencing of DNA molecules is performed, which has the characteristics of large throughput, small bias, and complete information. Therefore, NGS testing has become an important tool for disease diagnosis and scientific research. In the process of disease diagnosis, NGS detection is mainly divided into two aspects: DNA detection and RNA detection. DNA NGS detection is mainly used to analyze single nucleotide variants (Single nucleotide variants, SNVs), insertion or deletion mutations (Insertions and Deleti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2525/173C12Q2525/191C12Q2521/107C12Q2521/501C12Q2535/122
Inventor 江翱陈晶晶刘娜秦雪梅罗秉轮卢瑶王嫚曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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