CN117661123A - Single-cell library construction method for multiple sample mixing - Google Patents

Abstract

The application relates to the technical field of bioinformatics and discloses a multi-sample mixed single-cell library construction method, wherein genomic DNA of each sample is broken, and DNA fragments with bands distributed in 500bp are obtained; adding an adhesive primer combined with an adhesive tail end into the obtained DNA fragment, and adding a tail end leveling reagent for mixing reaction; designing an initial primer according to the DNA fragment, and respectively connecting a plurality of labels with the designed initial primer to obtain a fusion primer; connecting the first fusion primer with the head end of the DNA fragment by adopting an A joint, and connecting the second fusion primer with the tail end of the DNA fragment by adopting a P joint; purifying magnetic beads; performing PCR amplification on DNA fragments with different fusion primers at the head and the tail; and (3) after quantitative and sample mixing, separating and purifying by agarose gel to obtain a single-cell sequencing document. The method and the device are beneficial to reducing cost and have higher reliability.

Description

A multi-sample mixed single cell library construction method

Technical field

This application relates to the technical field of bioinformatics, specifically a multi-sample mixing single cell library construction method.

Background technique

Single-cell sequencing is a very costly method, so using mixed samples for sequencing can effectively reduce costs. However, the existing mixed sample solution can only mix up to 12 samples at a time. At the same time, due to cell conditions, adding more tags is not conducive to the activity of the samples and is difficult to distinguish. Therefore, a reliable mixed sample sequencing technology is urgently needed to solve these problems. .

Contents of the invention

The purpose of this application is to provide a multi-sample mixed single cell library construction method to solve the technical problems raised in the above background art.

In order to achieve the above objectives, this application discloses the following technical solutions:

A multi-sample mixed single cell library construction method, which includes the following steps in sequence:

Fragment the genomic DNA of each sample to obtain DNA fragments with concentrated bands distributed at 500bp;

Add sticky primers that bind to sticky ends to the obtained DNA fragments, and add end-filling reagents for mixed reactions;

Design initial primers based on DNA fragments, and connect several tags to the designed initial primers to obtain fusion primers;

Use an A linker to connect the first fusion primer to the first end of the DNA fragment, and use a P linker to connect the second fusion primer to the end of the DNA fragment;

Magnetic bead purification;

Perform PCR amplification of DNA fragments with different fusion primers at the beginning and end;

After quantification and mixing, agarose gel separation and purification are performed to obtain a single-cell sequencing library.

Preferably, the fusion primer includes an upstream universal primer sequence arranged sequentially along the 5' to 3' direction and a specific upstream primer sequence designed according to the target amplification region in the purified DNA fragment, and a sequence along the 5' to 3' direction. The downstream universal primer sequence and the specific downstream primer sequence designed according to the target amplification region in the purified DNA fragment are arranged in sequence.

Preferably, the upstream universal primer sequence and the downstream universal primer sequence are 16S rRNA sequencing primers respectively.

Preferably, the magnetic bead purification includes: using 1.2 times AMPure XP magnetic beads to purify the DNA fragments to remove excess small fragment adapters and primers in the DNA fragments.

Preferably, the A linker and P linker are linkers of Ion Torrent, Ion PGM, IonProton or Ion S5/S5XL sequencing platform.

Preferably, the concentration of the genomic DNA is 0.1-150ng/μL.

Preferably, the method further includes: a biological information analysis method based on library construction structure design matching;

The biological information analysis method includes:

Obtain sequencing data of the DNA sequence to be tested;

Input the sequencing data into the purification model and perform purification according to preset filtering rules to obtain purified sequencing data that retains only biological information;

Filter the purified sequencing data according to the preset tag filtering rules to obtain the characterization sequence corresponding to the purified sequencing data;

Compare the characterization sequence with preset tag sequence data to obtain fusion primer information;

Conduct GO enrichment analysis, KO enrichment analysis and WGS analysis based on the fusion primer information to obtain the sequence type, sequence distribution, and data base distribution characteristics corresponding to the characterization sequence;

The purified sequencing data, sequence types, sequence distribution, and data base distribution characteristics are statistically output as bioinformatics analysis results.

Preferably, the WGS analysis is implemented by using one or more of Refseq, GEO Database and Expression Atlas.

Beneficial effects: The multi-sample mixed single-cell library construction method of this application adopts the form of fusion primers, which enriches the number of tags, and the corresponding tags are connected to the head or end of the DNA fragment using adapters, thereby facilitating analysis and having It is beneficial to maintain the state of cells, easily distinguish cells and reduce the impact on cell activity. Furthermore, through later data filtering, it is easy to restore the true situation of the sample and quickly obtain relevant biological information.

Description of drawings

In order to explain the embodiments of the present application or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic flowchart of the multi-sample mixing single cell library construction method provided by the embodiment of the present application.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of this application.

As used herein, the term "comprising" is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus including a list of elements includes not only those elements but also other elements not expressly listed, or may also include Elements inherent to such a process, method, article, or equipment. Without further limitation, an element defined by the statement "comprising..." does not exclude the presence of additional identical elements in a process, method, article, or device that includes the stated element.

The applicant found that the existing mixing sample solution can only mix up to 12 samples at a time. We can mix more while maintaining the sample quality, and the suspension quality meets the requirements of various single cell platforms at the same time. Blood samples are particularly suitable and can significantly reduce costs.

Based on the above, this embodiment discloses a multi-sample mixed single cell library construction method as shown in Figure 1. The method includes the following steps in sequence:

S101-Fragment the genomic DNA of each sample to obtain DNA fragments concentrated at 500 bp; in this example, the concentration of the genomic DNA is 0.1-150ng/μL;

S102-Add sticky primers that bind to sticky ends to the obtained DNA fragments, and add end-filling reagents for mixed reactions;

S103-Design initial primers based on DNA fragments, and connect several tags to the designed initial primers to obtain fusion primers;

S104-Use A linker to connect the first fusion primer to the first end of the DNA fragment, and use P linker to connect the second fusion primer to the end of the DNA fragment;

S105-magnetic bead purification;

S106-Perform PCR amplification of DNA fragments with different fusion primers at the beginning and end;

S107-After quantification and sample mixing, perform agarose gel separation and purification to obtain a single-cell sequencing library.

In this embodiment, the fusion primer includes an upstream universal primer sequence arranged sequentially along the 5' to 3' direction and a specific upstream primer sequence designed according to the target amplification region in the purified DNA fragment, and along the 5' The downstream universal primer sequence and the specific downstream primer sequence designed according to the target amplification region in the purified DNA fragment are arranged sequentially in the 3' direction. Wherein, the upstream universal primer sequence and the downstream universal primer sequence are respectively 16S rRNA sequencing primers.

In this example, the magnetic bead purification includes: using 1.2 times AMPure XP magnetic beads to purify the DNA fragments to remove excess small fragment adapters and primers in the DNA fragments.

Possibly, the A linker and P linker are linkers of Ion Torrent, Ion PGM, IonProton or Ion S5/S5XL sequencing platform. It should be noted that Ion Torrent, Ion PGM, IonProton or Ion S5/S5XL sequencing platforms are commonly used sequencing platforms. In order to make the library more compatible, this embodiment uses adapters from commonly used sequencing platforms.

As a preferred implementation of this embodiment, the method further includes: a biological information analysis method based on library construction structure design matching;

The biological information analysis method includes:

Obtain sequencing data of the DNA sequence to be tested;

Input the sequencing data into the purification model and perform purification according to preset filtering rules to obtain purified sequencing data that retains only biological information;

Filter the purified sequencing data according to the preset tag filtering rules to obtain the characterization sequence corresponding to the purified sequencing data;

Compare the characterization sequence with preset tag sequence data to obtain fusion primer information;

Conduct GO enrichment analysis, KO enrichment analysis and WGS analysis based on the fusion primer information to obtain the sequence type, sequence distribution, and data base distribution characteristics corresponding to the characterization sequence;

The purified sequencing data, sequence type, sequence distribution, and data base distribution characteristics statistics are output as the biological information analysis results. Among them, the true situation of the sample is the difference between the purified sequencing data and the characterized sequence.

In this embodiment, the WGS analysis is implemented by using one or more of Refseq, GEO Database and Expression Atlas.

The multi-sample mixed single-cell library construction method described in this example adopts the form of fusion primers, which enriches the number of tags. Moreover, the corresponding tags are connected to the head or end of the DNA fragment using a linker, which facilitates analysis and is beneficial. Maintain cell status, easily distinguish cells and reduce impact on cell activity. Furthermore, through later data filtering, it is easy to restore the true situation of the sample and quickly obtain relevant biological information.

Finally, it should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or equivalent substitutions may be made to some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application shall be included in within the protection scope of this application.

Claims (8)

1. A multi-sample mixed single cell library construction method, characterized in that the method includes the following steps in sequence: Fragment the genomic DNA of each sample to obtain DNA fragments with concentrated bands distributed at 500bp; Add sticky primers that bind to sticky ends to the obtained DNA fragments, and add end-filling reagents for mixed reactions; Design initial primers based on DNA fragments, and connect several tags to the designed initial primers to obtain fusion primers; Use an A linker to connect the first fusion primer to the first end of the DNA fragment, and use a P linker to connect the second fusion primer to the end of the DNA fragment; Magnetic bead purification; Perform PCR amplification of DNA fragments with different fusion primers at the beginning and end; After quantification and mixing, agarose gel separation and purification are performed to obtain a single-cell sequencing library. 2. The multi-sample mixed single cell library construction method according to claim 1, characterized in that the fusion primer includes an upstream universal primer sequence arranged sequentially along the 5' to 3' direction and the purified DNA fragment according to The specific upstream primer sequence designed for the target amplification region, and the downstream universal primer sequence arranged sequentially along the 5' to 3' direction and the specific downstream primer sequence designed according to the target amplification region in the purified DNA fragment. 3. The multi-sample mixed single cell library construction method according to claim 2, characterized in that the upstream universal primer sequence and the downstream universal primer sequence are respectively 16S rRNA sequencing primers. 4. The multi-sample mixed single cell library construction method according to claim 1, characterized in that the magnetic bead purification includes: using 1.2 times AMPure XP magnetic beads to purify the DNA fragments to remove excess in the DNA fragments. small fragment adapters and primers. 5. The multi-sample mixed single cell library construction method according to claim 1, characterized in that the A linker and P linker are linkers of Ion Torrent, Ion PGM, IonProton or Ion S5/S5XL sequencing platform. 6. The multi-sample mixed single cell library construction method according to claim 1, characterized in that the concentration of the genomic DNA is 0.1-150ng/μL. 7. The multi-sample mixed single cell library construction method according to claim 1, characterized in that the method further includes: a biological information analysis method based on library construction structure design matching; The biological information analysis method includes: Obtain sequencing data of the DNA sequence to be tested; Input the sequencing data into the purification model and perform purification according to preset filtering rules to obtain purified sequencing data that retains only biological information; Filter the purified sequencing data according to the preset tag filtering rules to obtain the characterization sequence corresponding to the purified sequencing data; Compare the characterization sequence with preset tag sequence data to obtain fusion primer information; Conduct GO enrichment analysis, KO enrichment analysis and WGS analysis based on the fusion primer information to obtain the sequence type, sequence distribution, and data base distribution characteristics corresponding to the characterization sequence; The purified sequencing data, sequence types, sequence distribution, and data base distribution characteristics are statistically output as bioinformatics analysis results. 8. The multi-sample mixed single cell library construction method according to claim 7, characterized in that the WGS analysis is implemented by using one or more of Refseq, GEO Database and Expression Atlas.