阅读说明：本技术 胎儿游离dna的富集方法 (Method for enriching free DNA of fetus ) 是由 赵军 方楠 林少航 贾晋红 王伟伟 伍启熹 王建伟 刘倩 唐宇 于 2019-11-29 设计创作，主要内容包括：本发明提供了一种胎儿游离DNA的富集方法。该富集方法包括：采用20～33bp的Y型接头与游离DNA进行连接,得到总游离DNA的连接片段；采用磁珠对总游离DNA的连接片段进行纯化并回收50-150bp的游离DNA对应的连接片段,得到富集的胎儿游离DNA。该富集方法针对游离DNA中胎儿游离DNA集中在50-150bp这一特点,通过对二代测序文库构建过程中的操作步骤进行调整,对接头进行截短,并去除大于150bp的游离DNA片段,富集50-150bp的游离DNA片段,从而实现对于胎儿游离DNA的富集。(The invention provides a method for enriching free DNA of a fetus. The enrichment method comprises the following steps: connecting the free DNA with a Y-shaped joint of 20-33bp to obtain a connecting fragment of the total free DNA; purifying the connecting fragment of the total free DNA by adopting magnetic beads, and recovering the connecting fragment corresponding to the free DNA with the length of 50-150bp to obtain the enriched fetal free DNA. Aiming at the characteristic that the fetal free DNA in the free DNA is concentrated in 50-150bp, the enrichment method realizes the enrichment of the fetal free DNA by adjusting the operation steps in the construction process of the second-generation sequencing library, truncating the joint, removing the free DNA fragments larger than 150bp and enriching the free DNA fragments of 50-150 bp.)

1. A method for enriching fetal free DNA, the method comprising:

connecting the free DNA with a Y-shaped joint of 20-33bp to obtain a connecting fragment of the total free DNA;

and purifying the connecting fragments of the total free DNA by using magnetic beads, and recovering the connecting fragments corresponding to the free DNA with the length of 50-150bp to obtain the enriched fetal free DNA.

2. The enrichment method according to claim 1, wherein purifying the ligated fragments of the total free DNA and recovering the ligated fragments corresponding to 50-150bp of the free DNA to obtain the enriched fetal free DNA comprises:

adding magnetic beads with the volume of 0.8-1.2 times that of the connection fragments into the connection fragments of the total free DNA to remove the connection fragments corresponding to free DNA with the length of more than 150bp to obtain first purified fragments;

adding the magnetic beads and isopropanol into the first purified fragments, and controlling the total amount of the two times of adding the magnetic beads to reach 1.2-1.8 times of the volume of the first purified fragments, and the volume of the added isopropanol to be 0.2-2.0 times, preferably 0.5-0.9 times of the volume of the first purified fragments, thereby recovering the connecting fragments corresponding to the free DNA of 50-150bp, and obtaining the enriched fetal free DNA.

3. The enrichment method according to claim 1, wherein purifying the ligated fragments and recovering the ligated fragments corresponding to 50-150bp of the free DNA to obtain the enriched fetal free DNA comprises:

completely recovering the connecting segments of the total free DNA to obtain a recovered product;

removing the connecting fragment corresponding to the free DNA with the length of more than 150bp in the recovered product to obtain a second purified fragment;

recovering the connection fragment corresponding to the free DNA of 50-150bp in the second purified fragment to obtain the enriched fetal free DNA.

4. The enrichment method according to claim 3, wherein the total recovery of the ligated fragments of the total free DNA to obtain the recovered product comprises:

the recovery product is obtained by adding 1.0 to 2.0 times, preferably 1.2 to 1.8 times, the volume of the ligated fragments of the total free DNA to the ligated fragments of the total free DNA, and 0.2 to 2.0 times, preferably 0.5 to 1.0 times, the volume of the ligated fragments of the total free DNA to isopropanol, thereby completely recovering the ligated fragments of the total free DNA.

5. The enrichment method according to claim 3, wherein the removing of the ligated fragments corresponding to the free DNA larger than 150bp from the recovered product to obtain the second purified fragment comprises:

and adding 1.0-1.5 times, preferably 1.1-1.2 times of the volume of the magnetic beads of the recovered product to the recovered product so as to remove the connecting fragment corresponding to the free DNA with the length of more than 150bp, thereby obtaining the second purified fragment.

6. The enrichment method of claim 5, wherein recovering the ligated fragments corresponding to 50-150bp of the episomal DNA from the second purified fragment, and obtaining the enriched fetal episomal DNA comprises:

adding the magnetic beads into the second purification fragment again, and controlling the total volume of the magnetic beads to be 1.5-2.0 times, preferably 1.7-1.9 times of the volume of the second purification fragment;

and then adding 0.2-2.0 times, preferably 0.5-0.9 times of isopropanol of the volume of the second purified fragment or 0.5-4.0 times of absolute ethanol of the volume of the second purified fragment to complete the recovery of the connected fragment corresponding to the free DNA of 50-150bp, thereby obtaining the enriched free DNA of the fetus.

7. The enrichment method according to any one of claims 1 to 6, further comprising: and performing PCR amplification on the enriched fetal free DNA to construct a fetal free DNA library.

8. The enrichment method according to claim 7,

carrying out PCR amplification on the enriched fetal free DNA by adopting an amplification primer with index at one end to obtain an amplification library;

and purifying and recovering the amplified library to obtain the fetal free DNA library.

9. The enrichment method of claim 8, wherein the purifying and recovering the amplified library comprises:

purifying and recovering the amplified library by using 1.0-2.0 times, preferably 1.7-1.9 times of magnetic beads and 0.2-2.0 times, preferably 0.5-0.9 times of isopropanol of the amplified library volume; or

Purifying and recovering the amplified library by using 1.0-2.0 times, preferably 1.7-1.9 times of magnetic beads and 0.5-4.0 times of absolute ethyl alcohol.

10. The enrichment method according to any one of claims 1 to 6, wherein the 20-33bp Y-linker is an index sequence-free Y-linker of the Illumina sequencing platform.

Technical Field

The invention relates to the field of prenatal screening, in particular to a fetal free DNA enrichment method.

Background

Prenatal screening is of great significance for reducing birth defects. It can screen not only 21, 18 and 13 chromosome trisomy, but also diseases caused by chromosome aneuploidy (microdeletion, microduplication). In addition, it is also of great interest to contribute to the prevention of monogenic diseases (rare diseases). The method has the advantages that the proportion of the fetal free DNA in the total free DNA is increased, and the method has important significance for improving the accuracy of prenatal screening.

There are many existing methods for enriching fetal free DNA in peripheral blood, and the following categories are summarized:

the first type: enrichment of fetal DNA by isolation of nucleated red blood cells

Adult red blood cells are cell nucleus-free and do not contain DNA. Fetal red blood cells contain nuclei and present genomic DNA sequences. Enrichment of fetal free DNA can be achieved by separating nucleated red blood cells from maternal blood.

The limitations of this approach are: 1. the technical requirement is high; 2. the sample volume requirement is large, and the pregnant woman is difficult to accept; 3. the separation of nucleated red blood cells is long, and automation is difficult to realize; 4. the success rate is low; 5. the cost is high.

The second type: enrichment of cffDNA by methylation

Fetal free DNA contains more methylation modifications than adult. Fetal free DNA can be enriched by capture of methylated DNA.

The limitations of this approach are: 1. the technology is complicated and difficult; 2. the cost of screening free DNA of the fetus is high; 3. it is difficult to automate the experiment.

In the third category: screening protocol based on cffDNA size

The peak value of the cfDNA from the mother is 160-170bp, and the proportion of the free DNA from the fetus is higher in the free DNA smaller than 150 bp. By screening the free DNA smaller than 150bp, the proportion of the fetal free DNA in the total free DNA can be increased, thereby realizing the enrichment of the fetal free DNA.

Two protocols are commonly used to screen for smaller free DNA.

1. Carrying out agarose electrophoresis on the free DNA or the free DNA library, and then screening the free DNA fragments smaller than 150bp by cutting gel;

2. screening free DNA or a free DNA library by selectively purifying and recovering magnetic beads, removing the free DNA library with the length of more than 150bp, and recovering smaller free DNA fragments;

the limitations of such screening protocols are:

1. when the DNA fragments are screened and recovered through gel cutting recovery, the judgment of the DNA size is inaccurate, and the technical repeatability is poor;

2. when free DNA or libraries are screened by selectively purifying and recovering magnetic beads, a large amount of connector dimer pollution is generated in an Illumina sequencing library building system, and sequencing quality and effective utilization rate of reads are reduced.

In the field of second-generation sequencing at present, the market proportion of the illumina sequencing instrument is more than 70 percent, and the illumina sequencing instrument is in the dominating position. Library construction based on the Illumina sequencer yielded linker dimers 120bp in length. In fetal free DNA enrichment, small free DNA fragments (50-150bp) need to be recovered, and after a 120bp linker is added, a library fragment of 170bp and a linker dimer of 120bp are difficult to distinguish by using a selective purification magnetic bead, so that a large amount of linker dimers or small free DNA fragments remain in the library, and the library cannot be sufficiently enriched.

In conclusion, the existing enrichment method has the defect of low efficiency.

Disclosure of Invention

The invention mainly aims to provide a method for enriching free DNA of a fetus, which aims to solve the problem of low enrichment efficiency of free DNA of the fetus in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a method for enriching fetal free DNA, the method comprising: connecting the free DNA with a Y-shaped joint of 20-33bp to obtain a connecting fragment of the total free DNA; purifying the connecting fragment of the total free DNA by adopting magnetic beads, and recovering the connecting fragment corresponding to the free DNA with the length of 50-150bp to obtain the enriched fetal free DNA.

Further, purifying the ligated fragments of total free DNA and recovering the ligated fragments corresponding to 50-150bp of free DNA to obtain enriched fetal free DNA comprising: adding magnetic beads with the volume of 0.8-1.2 times that of the connecting fragments into the connecting fragments of the total free DNA to remove the connecting fragments corresponding to the free DNA with the length of more than 150bp to obtain first purified fragments; adding magnetic beads and isopropanol into the first purified fragment, and controlling the total amount of the two times of magnetic beads to reach 1.2-1.8 times of the volume of the first purified fragment, wherein the volume of the added isopropanol is 0.2-2.0 times, preferably 0.5-0.9 times of the volume of the first purified fragment, thereby recovering a connecting fragment corresponding to 50-150bp of free DNA and obtaining the enriched fetal free DNA.

Further, purifying the ligated fragments and recovering the ligated fragments corresponding to 50-150bp of the free DNA to obtain enriched fetal free DNA comprises: completely recovering the connecting segments of the total free DNA to obtain a recovered product; removing the connecting fragment corresponding to the free DNA larger than 150bp in the recovered product to obtain a second purified fragment; recovering the connection fragment corresponding to the free DNA of 50-150bp in the second purified fragment to obtain the enriched fetal free DNA.

Further, the whole recovery of the ligated fragments of total free DNA to obtain the recovered product comprises: the total recovery of the ligated fragments of total free DNA is carried out by adding 1.0 to 2.0 times, preferably 1.2 to 1.8 times, the volume of the ligated fragments of total free DNA and 0.2 to 2.0 times, preferably 0.5 to 1.0 times, the volume of isopropanol to the ligated fragments of total free DNA to thereby obtain a recovered product.

Further, removing the ligated fragment corresponding to the free DNA larger than 150bp in the recovered product to obtain a second purified fragment comprises: the second purified fragment is obtained by adding 1.0-1.5 times, preferably 1.1-1.2 times, volume of magnetic beads to the recovered product to remove the ligated fragment corresponding to free DNA larger than 150 bp.

Further, recovering the ligated fragments corresponding to 50-150bp of the free DNA in the second purified fragment to obtain enriched fetal free DNA comprises: adding the magnetic beads into the second purified fragment again, and controlling the total volume of the magnetic beads to be 1.5-2.0 times, preferably 1.7-1.9 times of the volume of the second purified fragment; and then adding isopropanol with the volume of 0.2-2.0 times, preferably 0.5-0.9 times, or absolute ethanol with the volume of 0.5-4.0 times, of the second purified fragment to complete the recovery of the connected fragment corresponding to the free DNA with the length of 50-150bp, so as to obtain the enriched free DNA of the fetus.

Further, the enrichment method further comprises: and performing PCR amplification on the enriched fetal free DNA to construct a fetal free DNA library.

Further, carrying out PCR amplification on the enriched fetal free DNA by adopting an amplification primer with index at one end to obtain an amplification library; and purifying and recovering the amplified library to obtain a fetal free DNA library.

Further, the purification and recovery of the amplified library comprises: purifying and recovering the amplified library by using 1.0-2.0 times, preferably 1.7-1.9 times of magnetic beads and 0.2-2.0 times, preferably 0.5-0.9 times of isopropanol of the amplified library; or purifying and recovering the amplified library by using 1.0-2.0 times, preferably 1.7-1.9 times of magnetic beads and 0.5-4.0 times of absolute ethyl alcohol.

Furthermore, the Y-type joint of 20-33bp is a Y-type joint without an index sequence of the Illumina sequencing platform.

By applying the technical scheme of the invention, aiming at the characteristic that the fetal free DNA in the free DNA is concentrated in 50-150bp, the enrichment method realizes the enrichment of the fetal free DNA by adjusting the operation method in the construction process of the second-generation sequencing library, truncating the joint, removing the free DNA fragments larger than 150bp and enriching the free DNA fragments of 50-150 bp.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a flow chart for enrichment of fetal free DNA during prenatal diagnosis using the general library construction method:

FIG. 2 shows a flow chart of the improved banking process of the present application for enrichment of fetal free DNA:

FIG. 3 shows the sequence of the Y-linker and a schematic structural diagram thereof in the method for enrichment of fetal free DNA by Illumina sequencing platform;

FIGS. 4A to 4H are graphs showing the DNA distribution ranges obtained by the different recovery schemes in example 1 of the present application;

FIGS. 5A to 5B are graphs showing the distribution range of DNA obtained by pooling the full-length adaptor (FIG. 5A) and the short adaptor (FIG. 5B) in example 2 of the present application;

FIGS. 6A to 6D are graphs showing the distribution of DNA fragments in the library obtained by different selective purification recovery schemes in example 3 of the present application;

FIGS. 7A to 7F are graphs showing the distribution of DNA fragments in the library obtained by different selective purification recovery schemes in example 4 of the present application;

FIG. 8 shows the concentration of fetal free DNA obtained from the sequencing and data analysis of 38 libraries obtained by the enrichment method of the present application and the existing general library construction method in example 5 of the present application.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

As mentioned in the background, the peak of free DNA extracted from plasma of pregnant women was found to be between 160 and 170bp by capillary electrophoresis detection (Agilent 2100 or similar instrument). The extracted free DNA contains free DNA of both mother origin and fetal origin. Compared with maternal-derived free DNA, fetal-derived free DNA is smaller and has a distribution range mainly between 60-150 bp. In the prior art, as shown in fig. 1, a method for enriching fetal-derived free DNA in free DNA mainly utilizes a general library construction process to enrich, and performs purification and recovery (mainly magnetic bead purification and recovery) after adaptor connection and PCR amplification.

In the current enrichment protocol, researchers typically focus only on the main 160-170bp peak of free DNA. In the Illumina sequencing system, the main peak after free DNA ligation to the linker is around 290 bp. The Illumina sequencing system usually generates linker dimer with the length of 120bp in the library building process, and the existence of the linker dimer can cause great influence on the sequencing effect. Since the 120bp linker dimer differs in length from the 290bp library main peak, the linker dimer can be selectively removed by selective purification of the beads.

In the present application, in order to increase the proportion of fetal free DNA in total free DNA, it is necessary to remove free DNA fragments having a length of more than 150bp and recover free DNA fragments of 50-150 bp. However, it was found in the experiments: the length of the free DNA of 50bp after being connected with the linker is 170bp, and the length of the linker dimer is 120bp, so that the linker dimer is difficult to remove under the condition of not losing the free DNA fragment of 50bp when the magnetic beads are used for purification.

In order to recover as much as possible smaller free DNA fragments of 50-150bp while removing the linker dimer, the inventors tried to replace the original full-length linker with a short linker of 20-33bp in length (as shown in FIG. 3). Although the length of the linker dimer (40-66bp) formed by the short linker is the same as that of the short free DNA fragment (90-110bp) connected with the linker by 50bp, the shorter the fragment is, the more difficult the fragment is to recover, so that the two fragments can be better distinguished by selectively purifying and recovering magnetic beads, and a library without obvious linker contamination can be obtained.

When selective purification magnetic beads (AMPure XP or similar products) are used for DNA fragment purification and recovery, generally, the smaller the DNA fragment is, the larger the amount of the magnetic beads is required to be used; the smaller the DNA fragment, the lower the recovery efficiency under the same reagent amount. Further, in order to improve the recovery efficiency of small fragment DNA (mainly 50-150bp), isopropanol was added to the recovery volume in an amount of 0.2-2.0 (preferably 0.5-1.0) times the sample volume.

Further, the inventor researches and also finds that: in the library construction process, after the short linker is connected, firstly, the DNA of the ligation product is fully purified and recovered (for example, 1.8 times of the volume of purified magnetic beads and 0.7 times of the volume of isopropanol are added), then, the recovered product is selectively purified and recovered, large fragments (for example, 1.0-1.5 times of the volume of purified magnetic beads are added) are removed, and small fragments (for example, purified magnetic beads which are complementary to 1.8 times of the volume of a sample and 0.7 times of the volume of isopropanol are recovered), so that not only can the stability of selective recovery and purification be improved, but also the universality of the improved fetal free DNA enrichment scheme can be improved.

On the basis of the above research results, the applicant proposed the technical solution of the present application. In an exemplary embodiment of the present application, there is provided a method for enrichment of fetal free DNA, the method comprising: connecting the free DNA with a Y-shaped joint of 20-33bp to obtain a connecting fragment of the total free DNA; purifying the connecting fragment of the total free DNA by adopting magnetic beads, and recovering the connecting fragment corresponding to the free DNA with the length of 50-150bp to obtain the enriched fetal free DNA.

In a preferred embodiment, as shown in FIG. 2, the purification of the ligated fragments of total free DNA using magnetic beads and the recovery of the ligated fragments corresponding to 50-150bp of free DNA, and obtaining enriched fetal free DNA comprises: adding magnetic beads with the volume of 0.8-1.2 times that of the connecting fragments into the connecting fragments of the total free DNA to remove the connecting fragments with the length more than 150bp corresponding to the free DNA to obtain first purified fragments; adding magnetic beads and isopropanol into the first purified fragment, and controlling the total amount of the two times of magnetic beads to reach 1.2-1.8 times of the volume of the first purified fragment, wherein the volume of the added isopropanol is 0.2-2.0 times, preferably 0.5-0.9 times of the volume of the first purified fragment, thereby recovering a connecting fragment corresponding to 50-150bp of free DNA and obtaining the enriched fetal free DNA.

In a preferred embodiment, as shown in FIG. 2, the purification of the ligated fragments using magnetic beads and the recovery of ligated fragments of 50-150bp of free DNA to obtain enriched fetal free DNA comprises: completely recovering the ligation products of the total free DNA to obtain recovered products; removing the connecting fragment corresponding to the free DNA larger than 150bp in the recovered product to obtain a second purified fragment; recovering the connection fragment corresponding to the free DNA of 50-150bp in the second purified fragment to obtain the enriched fetal free DNA.

In a preferred embodiment, the total recovery of the ligated fragments of total free DNA to obtain a recovered product comprises: the total recovery of the ligated fragments of total free DNA is carried out by adding 1.0 to 2.0 times, preferably 1.2 to 1.8 times, the volume of the ligated fragments of total free DNA and 0.2 to 2.0 times, preferably 0.5 to 1.0 times, the volume of isopropanol to the ligated fragments of total free DNA to thereby obtain a recovered product.

In a preferred embodiment, removing the ligated fragments corresponding to the free DNA larger than 150bp from the recovered product to obtain a second purified fragment comprises: a second purified fragment is obtained by adding 1.0 to 1.5 times, preferably 1.1 to 1.2 times, the volume of the magnetic bead to the recovered product, thereby selectively removing a ligated fragment corresponding to a free DNA larger than 150 bp.

In a preferred embodiment, recovering the ligated fragments corresponding to 50-150bp of free DNA in the second purified fragment to obtain enriched fetal free DNA comprises: adding the magnetic beads into the second purified fragment again, and controlling the total volume of the magnetic beads to be 1.5-2.0 times, preferably 1.7-1.9 times of the volume of the second purified fragment; and then adding isopropanol with the volume of 0.2-2.0 times, preferably 0.5-0.9 times, or absolute ethanol with the volume of 0.5-4.0 times, of the second purified fragment to complete the recovery of the connecting fragment corresponding to the free DNA with the length of 50-150bp, so as to obtain the enriched free DNA of the fetus.

In a preferred embodiment, as shown in fig. 2, the above enrichment method further comprises: and performing PCR amplification on the enriched fetal free DNA to construct a fetal free DNA library.

In a preferred embodiment, as shown in FIG. 2, PCR amplification is performed on the enriched fetal-free DNA using amplification primers with index at one end to obtain an amplification library; and purifying and recovering the amplified library to obtain a fetal free DNA library.

In a preferred embodiment, the purification and recovery of the amplified library comprises: purifying and recovering the amplified library by using 1.0-2.0 times, preferably 1.7-1.9 times of magnetic beads and 0.2-2.0 times, preferably 0.5-0.9 times of isopropanol of the amplified library; or purifying and recovering the amplified library by using 1.0-2.0 times, preferably 1.7-1.9 times of magnetic beads and 0.5-4.0 times of absolute ethyl alcohol.

In a preferred embodiment, the 20-33bp Y-type linker is a Y-type linker without index sequence of the Illumina sequencing platform.

In the above step of linker ligation, the linker without tag sequences such as index or barcode is used, and it should be noted that the present application is not limited to library sequencing using the illumina platform.

In the enrichment method, the number of PCR amplification cycles is set reasonably according to actual needs. In a preferred embodiment of the present application, the fetal free DNA library is obtained by performing 1 to 15 rounds, preferably 5 to 12 rounds of PCR amplification.

In a second exemplary embodiment of the present application, a fetal episomal DNA library is provided, which is constructed by the enrichment method described above. The free DNA fragments are connected by adopting a short joint, and then the free DNA fragments after the joint is connected are selectively purified and recovered by adopting a selective purification and recovery magnetic bead (containing isopropanol). Because the short linker forms a linker dimer with a shorter length, the linker dimer is less easily recovered when the bead-selective purification recovery is used, and accordingly, the length of the free DNA connected to the short linker is only 50bp, but the free DNA is more easily recovered when the bead-selective purification recovery is used. Further avoiding the pollution of the linker dimer to a certain extent and realizing the effective enrichment of free DNA of the fetus. Namely, the enrichment degree of the target fragment of the fetal free DNA in the library constructed by the method is high, and the effective data rate in the data obtained after sequencing is also high.

The advantageous effects of the present application will be further described with reference to specific examples.