CN110607563A - Sequencing library of filter paper sheet dry blood spots and construction method thereof - Google Patents
Sequencing library of filter paper sheet dry blood spots and construction method thereof Download PDFInfo
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Abstract
The invention provides a sequencing library of a filter paper sheet dried blood spot and a construction method thereof. The construction method comprises the following steps: placing the filter paper sheet dry blood spot sample in a full-automatic liquid treatment workstation for DNA extraction; and constructing a library by using the extracted DNA to obtain a sequencing library. The DNA extraction is carried out on the filter paper sheet dry blood spot sample by utilizing the full-automatic liquid treatment workstation, so that not only can the high-quality DNA extraction be realized, but also the sequencing library can be successfully constructed aiming at the filter paper sheet dry blood spot sample. The difficulty of constructing a library of the filter paper sheet dry blood spot sample at a low initial amount is solved, so that the trace sample can be sequenced by using high flux.
Description
Technical Field
The invention relates to the field of high-throughput sequencing library construction, in particular to a sequencing library of a filter paper sheet dry blood spot and a construction method thereof.
Background
The filter paper sheet is an excellent carrier for blood specimen collection and transport. The whole blood is dripped on a filter paper sheet to prepare filter paper sheet Dry Blood Spots (DBS), has good biological stability and safety, is convenient to store and transport, and is particularly suitable for sample collection, storage and transport in remote areas of developing countries.
At present, samples for second-generation sequencing gene detection mainly come from oral cells, saliva and peripheral blood of a detected person, but the methods inevitably encounter some problems in the process of collecting, storing and transporting the samples. Meanwhile, the whole blood needs to be transported by a special person in a cold chain due to the limitation of the transportation of biological dangerous goods. Especially when the screening scope is extended to remote, medical areas, difficulties are brought to the collection, storage and transportation of the examiners. Furthermore, since the total blood volume of a newborn is typically 250ml to 350ml, it is certainly very dangerous for the newborn if peripheral blood (5ml) is collected as required, and oral cells and saliva are difficult to obtain from the oral cavity of the infant, resulting in scarce sample sources. At present, the filter paper sheet dried blood spots are widely used with good biological stability and safety, but the filter paper sheet dried blood spots are mainly applied to tandem mass spectrometry or gene detection of single genetic diseases, and at present, few second-generation sequencing based on a liquid phase capture technology adopts a filter paper sheet dried blood spots to carry out gene detection of multiple genetic diseases.
The current filter paper sheet dry blood spots are usually used for detecting HIV type I DNA, so as to solve the practical difficulties existing in the whole blood sample collection, transportation and storage of HIV-1 infected persons. It only involves the detection of general DNA, and has low requirements on the concentration and purity of DNA, as long as general PCR can be performed. The specific operation is as follows: DNA extraction is carried out on the filter paper sheet dry blood spots, three pairs of highly conserved primers are designed aiming at env, gag and pol regions of HIV, and HIV-1DNA is amplified by nested PCR.
However, with the extensive development of molecular level research and the rapid development of high throughput sequencing technology in recent years, the extraction of high-yield and high-purity genomic DNA from peripheral blood or tissues for library construction to achieve high-throughput sequencing has become an important and relatively common experimental means. In view of the requirement of high-throughput sequencing library construction on DNA quantity, corresponding requirements are also provided for the extraction method and the extraction quality of DNA from different sources.
Although the first discovery in 1869 that nucleic acids are classified into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), the methods for extracting nucleic acids have been under improvement, such as the optimization and improvement of various experimental materials: sodium citrate, RNase, SDS, guanidine thiocyanate and the like are used for nucleic acid extraction in sequence. Some conventional methods such as CTAB method, Trizol method, lithium chloride method, etc. are still used in the existing nucleic acid extraction method. There are also emerging methods such as silica matrix methods, anion exchange methods, and nanobead extraction. Regardless of the method, the method mainly comprises 3 steps: 1) promoting cell rupture and releasing nucleic acid by physical or chemical method; 2) separating nucleic acid from substances such as proteins in the cell; 3) eluting and enriching the extracted nucleic acid.
Although all extraction methods have a communicating principle, the traditional nucleic acid extraction technology has complex extraction steps, takes long time and has low efficiency, the initial library building amount is at least 300ng, the DNA extraction and library building are both manual operations, the DNA extraction flux is 48 samples per day, the library building can be completed within 3 days, and the risk of artificial mixed sample is increased.
Moreover, although there are currently a few database construction methods for low initial amounts, there are mainly transposase method database construction and general procedure database construction. The method for constructing the library by the transposase method mainly comprises the steps of randomly cutting genome DNA by using the transposase, adding joints at two ends of a fragment, and then amplifying to construct the library, wherein the method is an efficient low-initial-amount (the initial amount of DNA is 60ng) library construction method. However, this method has a significant disadvantage in that it is susceptible to the interference of impurities in the sample during random cleavage disruption using transposase, resulting in failure of library construction. When the ordinary process adopts 80ng of initial amount of DNA for library construction, the special sample of the filter paper sheet dry blood spots has fewer samples, the DNA acquisition difficulty is high, and the extracted genomic DNA is less (less than or equal to 60ng), so the library construction is basically difficult according to the existing library construction process and method.
Therefore, the DNA library construction aiming at the filter paper sheet dry blood spot special sample has no corresponding solution in the prior art.
Disclosure of Invention
The invention mainly aims to provide a sequencing library of a filter paper sheet dry blood spot and a construction method thereof, and aims to solve the problem that the library construction of a filter paper sheet dry blood spot sample is difficult 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 constructing a sequencing library of dried blood spots of filter paper sheets, the method comprising: placing the filter paper sheet dry blood spot sample in a full-automatic liquid treatment workstation for DNA extraction; and constructing a library by using the extracted DNA to obtain a sequencing library.
Further, the step of placing the filter paper sheet dry blood spot sample in a full-automatic liquid processing workstation for DNA extraction comprises the following steps: placing the filter paper sheet dry blood spot sample in a pore plate for cracking; the well plate was placed in a fully automated liquid handling workstation for DNA extraction.
Further, before placing the filter paper sheet dry blood spot sample in the well plate for lysis, the construction method further comprises the following steps: punching the filter paper sheet dry blood spot sample by using a puncher to obtain a punched sample; placing the punched sample in a well plate; lysis was performed by adding lysis solution to the well plate.
Further, the diameter of the puncher is 6-12 mm, and a preferred pore plate is a 96 pore plate; preferably, the lysis is incubation at 50-60 ℃ for 15-45 min.
Further, the well plate is placed in a nucleic acid extraction chamber of a full-automatic liquid processing workstation, and a dry blood spot extraction mode is selected for DNA extraction.
Further, the library construction is performed by using the extracted DNA, and the step of obtaining a sequencing library comprises the following steps: and (3) constructing a library by using 60-80 ng of extracted DNA to obtain a sequencing library.
Further, library construction includes: breaking 60-80 ng of the extracted DNA to obtain fragment DNA; carrying out end repair and adding A on the fragment DNA to obtain a repair band A fragment; performing joint connection on the repair belt A segment to obtain a belt joint segment; and carrying out PCR amplification on the fragments with the connectors to obtain a sequencing library.
Further, breaking 60-80 ng of the extracted DNA, and carrying out electrophoresis gel purification on the broken DNA to obtain fragment DNA.
Further, PCR amplification is carried out on the tape joint fragments, and magnetic bead purification is carried out on products of the PCR amplification to obtain a sequencing library.
In order to achieve the above object, according to one aspect of the present invention, there is provided a sequencing library of dried blood spots of filter paper sheets, which is constructed by any one of the above-described construction methods.
By applying the technical scheme of the invention, the DNA extraction is carried out on the filter paper sheet dry blood spot sample by using the full-automatic liquid treatment workstation, so that not only can the high-quality (high-purity) DNA extraction be realized, but also the sequencing library can be successfully constructed aiming at the filter paper sheet dry blood spot sample. The difficulty of constructing a library of the filter paper sheet dry blood spot sample at a low initial amount is solved, so that the trace sample can be sequenced by using high flux.
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.
In order to make up for this gap, in a typical embodiment of the present application, there is provided a method for constructing a library for sequencing dried blood spots of filter paper sheets, the method comprising: placing the filter paper sheet dry blood spot sample in a full-automatic liquid treatment workstation for DNA extraction; and constructing a library by using the extracted DNA to obtain a sequencing library.
The DNA extraction is carried out on the filter paper sheet dry blood spot sample by utilizing the full-automatic liquid treatment workstation, so that not only can the high-quality (high-purity) DNA extraction be realized, but also the sequencing library can be successfully constructed aiming at the filter paper sheet dry blood spot sample. The difficulty of constructing a library of the filter paper sheet dry blood spot sample at a low initial amount is solved, so that the trace sample can be sequenced by using high flux.
Specifically, the step of placing the filter paper sheet dry blood spot sample in a full-automatic liquid treatment workstation for DNA extraction is carried out according to the operation instruction of the instrument. In a preferred embodiment, the step of placing the filter paper sheet dry blood spot sample in a fully automated liquid handling workstation for DNA extraction comprises: placing the filter paper sheet dry blood spot sample in a pore plate for cracking; the well plate was placed in a fully automated liquid handling workstation for DNA extraction.
The filter paper sheet dry blood spot sample can be placed in the pore plate in various ways, the dry blood spot can be directly cut into small fragments and placed in the pore plate, and other ways for enabling the pore plate to contain the dry blood spot can also be adopted. In a preferred embodiment, before placing the filter paper sheet dried blood spot sample in the well plate for lysis, the construction method further comprises: punching the filter paper sheet dry blood spot sample by using a puncher to obtain a punched sample; placing the punched sample in a well plate; lysis was performed by adding lysis solution to the well plate. The sampling area can be uniform by the perforator, and if the sample carrying amount of the dry blood spots is the same, the sample carrying amount of the sampling area is relatively uniform.
When the puncher is used for sampling, the diameter of the specifically selected puncher can be selected according to needs. In a preferred embodiment, the diameter of the hole puncher is 6-12 mm, and the preferred pore plate is a 96 pore plate; preferably, the lysis is incubation at 50-60 ℃ for 15-45 min. The cracking temperature and time can be adjusted according to the sample.
In a preferred embodiment, the well plate is placed in the nucleic acid extraction chamber of a fully automated liquid handling workstation and the dried blood spot extraction mode is selected for DNA extraction.
After the filter paper sheet dry blood spots are extracted by using a full-automatic liquid treatment workstation to obtain high-quality and high-purity DNA, the specific library building step can adopt a conventional library building method. In a preferred embodiment, library construction using the extracted DNA, and the step of obtaining a sequencing library comprises: and (3) constructing a library by using 60-80 ng of extracted DNA to obtain a sequencing library.
In a preferred embodiment, library construction comprises: breaking 60-80 ng of the extracted DNA to obtain fragment DNA; carrying out end repair and adding A on the fragment DNA to obtain a repair band A fragment; performing joint connection on the repair belt A segment to obtain a belt joint segment; and carrying out PCR amplification on the fragments with the connectors to obtain a sequencing library.
In a preferred embodiment, 60-80 ng of the extracted DNA is fragmented, and the fragmented DNA is subjected to electrophoresis gel purification to obtain fragment DNA.
In a preferred embodiment, the linker fragment is subjected to PCR amplification, and the products of the PCR amplification are subjected to magnetic bead purification to obtain a sequencing library.
In a second exemplary embodiment of the present application, a sequencing library of dried blood spots of filter paper sheets is provided, which is constructed by any one of the above-mentioned construction methods.
The advantageous effects of the present application will be explained in detail below with reference to specific examples. It should be noted that, in the following examples, reagents or consumables are available from Tiangen Biochemical technology Ltd unless otherwise specified.
The method comprises the following steps of (1) constructing a sequencing library by taking a whole peripheral blood filter paper sheet Dried Blood Spot (DBS) as an experimental material:
1. DNA extraction
1) The DBS with the diameter of 6mm is driven into a centrifugal tube with the volume of 1.5ml by a puncher, and the head of the puncher is sterilized by burning and alcohol wiping, so that cross contamination is avoided.
2) Adding 500ul of lysate into the centrifuge tube filled with the blood slices, and incubating for 0.5 hour at 56 ℃;
3) adding the liquid after incubation treatment into a 96-well plate, putting all samples into a nucleic acid extraction chamber after the addition is finished, selecting a dry blood spot extraction mode, and automatically finishing the extraction by an instrument.
2. 60ng initial quantity library construction
1) And (3) taking a human standard substance, diluting by 100 times, shaking, uniformly mixing, and accurately measuring the concentration by using Qbuit.
2) The volume required to add 60ng was calculated as the measured concentration.
3) Samples were ultrasonically disrupted using a Bioruptor instrument.
4) Detecting after the DNA is broken, carrying out dispensing and electrophoresis detection on the sample and the prepared marker, and purifying an electrophoresis strip when the electrophoresis strip is positioned at a position of 200bp-300 bp;
5) the end was repaired and A was added, and the reaction system is shown in Table 1 below.
Table 1:
| reagent | Volume of |
| DNA sample | 42μl |
| Buffer solution 1 | 6.8μl |
| Enzyme 1 | 1.2ul |
| General System | 50ul |
The reaction procedure for the repair plus a was as follows:
37℃ 30min，
72℃ 30min，
4℃ Hold。
6) connecting a joint: after the reaction was completed, the sample was placed on ice, and the reaction system was as shown in table 2 below:
table 2:
| reagent | Volume of |
| End-repair plus A product | 50ul |
| Buffer solution 2-1 | 8.4ul |
| Buffer 2-2 | 15ul |
| Enhancer | 1.6ul |
| Joint | 3ul |
| Enzyme 2 | 1ul |
| RNase-free water | 1ul |
| General System | 80ul |
The reaction procedure was as follows:
20℃ 30min。
7) purifying the connected product by M270 streptavidin magnetic beads, carrying out PCR amplification on the purified sample, and carrying out the following reaction procedures:
8) the amplified product was purified using M270 streptavidin magnetic beads:
9) library quantification, the concentration of the library is 5 ng/. mu.L (the concentration of the library should be more than or equal to 5 ng/. mu.L, otherwise the library is regarded as the construction failure).
10) Library hybridization: taking out a hybridization reagent and a blocking reagent, preparing a hybridization reaction system, concentrating a DNA library, adding a hybridization buffer solution and probes (the probes in the embodiment are a composition of 1282 probes for detecting 55 genes shown in Table 3, the length of a CDS region covered by UCSC is 101001bp, the coverage of a target region is 101184bp, the probes are designed and synthesized by IDT company, 1282 genes are counted after synthesis, and all capture regions are covered), and hybridizing overnight.
Table 3:
11) capture and elution of hybridized library: the hybridization library was captured with M270 streptavidin magnetic beads and PCR amplified to enrich it continuously.
12) The amplified DNA library was purified with M270 streptavidin magnetic beads:
13) and (4) accurately quantifying the concentration of the library by using the Qubit, diluting the library, and sending the library for inspection.
And (3) purifying the amplified product by using 1 time of M270 streptavidin magnetic beads, carrying out fragment sorting on the obtained purified product by using 0.7-0.85 time of M270 streptavidin magnetic beads, namely, reserving the supernatant when the 0.7 time of magnetic beads are purified, supplementing the supernatant to the 0.85 time of M270 streptavidin magnetic beads, and purifying to obtain the library with the size of 300-600 bp.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the invention reduces the initial amount of the constructed library to 60ng, and simultaneously adopts a large automatic liquid workstation to automatically complete the sample nucleic acid extraction work, compared with the traditional manual operation, the invention has the following advantages: 1) no cross contamination; 2) in each work of the automatic system, an operator only needs to do installation work and arrange the pore plates, and the whole extraction process does not need human participation, so that time and labor are saved; 3) the flux of the extracted sample is large, 384 parts per day, and the quality purity of the extracted nucleic acid is better. According to the invention, by optimizing the library construction process and method, the difficulty of low initial quantity library construction is solved, so that a trace sample can be sequenced by using high throughput.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for constructing a sequencing library of dried blood spots of filter paper sheets is characterized by comprising the following steps:
placing the filter paper sheet dry blood spot sample in a full-automatic liquid treatment workstation for DNA extraction;
and constructing a library by using the extracted DNA to obtain the sequencing library.
2. The method of constructing according to claim 1, wherein the step of placing the filter paper sheet dried blood spot sample in a fully automated liquid handling workstation for DNA extraction comprises:
placing the filter paper sheet dry blood spot sample in a pore plate for cracking;
placing the well plate in the fully automatic liquid handling workstation for DNA extraction.
3. The method of constructing according to claim 2, wherein before placing the filter paper sheet dried blood spot sample in a well plate for lysis, the method of constructing further comprises:
punching the filter paper sheet dry blood spot sample by using a puncher to obtain a punched sample;
placing the punched sample in the well plate;
adding a lysis solution to the well plate for the lysis.
4. The construction method according to claim 3, wherein the diameter of the punch is 6-12 mm,
preferably, the well plate is a 96 well plate;
preferably, the cracking is carried out at 50-60 ℃ for 15-45 min.
5. The construction method according to any one of claims 2 to 4, wherein the well plate is placed in a nucleic acid extraction chamber of the fully automated liquid handling workstation, and a dried blood spot extraction mode is selected for DNA extraction.
6. The method of any one of claims 1 to 4, wherein library construction is performed using the extracted DNA, and the step of obtaining the sequencing library comprises:
and constructing a library by using the DNA extracted by 60-80 ng to obtain the sequencing library.
7. The method of construction according to claim 6, wherein the library construction comprises:
breaking 60-80 ng of the extracted DNA to obtain fragment DNA;
carrying out end repair and adding A on the fragment DNA to obtain a repair band A fragment;
performing joint connection on the repair belt A segment to obtain a belt joint segment;
and carrying out PCR amplification on the fragments with the connectors to obtain the sequencing library.
8. The construction method according to claim 7, wherein the fragment DNA is obtained by fragmenting 60-80 ng of the extracted DNA and then subjecting the fragmented DNA to electrophoresis gel purification.
9. The method of claim 7, wherein the sequencing library is obtained by performing PCR amplification on the adaptor-ligated fragment and performing magnetic bead purification on the PCR-amplified product.
10. A sequencing library of dried blood spots on filter paper sheets, wherein the sequencing library is constructed by the construction method of any one of claims 1 to 9.
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Cited By (1)
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