CN109609597A - A kind of construction method of low initial amount PCR-free high-throughput sequencing library - Google Patents
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Abstract
The present invention relates to field of bioinformatics, specifically provide a kind of construction method of low initial amount PCR-free high-throughput sequencing library, the following steps are included: (1) extracts cfDNA in blood plasma, end reparation and 5 ' terminal phosphates are carried out to it, end is repaired product and recycled with magnetic beads for purifying；(2) product is repaired to end after purification, 3 ' ends add " A " base and connector interfaces system to be incorporated into a system to be reacted；(3) connector product magnetic beads for purifying obtains sequencing library.The advantages of construction method, is at low cost, time-consuming short, library construction is not necessarily to PCR amplification, keep stable data output, it is of less demanding to the amount of starting DNA, only the cfDNA dosage of 1-2ng, which just can satisfy, builds library needs, for being effectively sequenced, there is boundless application prospect in realizing the research such as high-flux sequence, non-invasive diagnosis.
Description
Technical field
The present invention relates to field of bioinformatics, specifically, it is mixed to be related to a kind of Multi-example suitable for high-flux sequence
Close the construction method of sequencing library.
Background technique
With the progress of science and technology, conventional capillary electrophoresis tube Sanger sequencing cannot fully meet science and grind
Study carefully, clinical application demand；Gene order-checking need flux is higher, efficiency faster, the lower technology platform of expense.High-flux sequence
(NGS, the also referred to as second generation sequencing) technology is come into being.The core concept of high throughput sequencing technologies be while synthesis while be sequenced (SBS,
Sequencing by synthesis), i.e., it is identified at once after one wheel base of synthesis every time, then synthesizes next round alkali again
Base.Existing technology platform mainly includes illumina/Hiseq, NextSeq, Hiseq X ten, NovaSeq and Life
Technologies system, PGM, Proton etc..Flux highest is using 6000 System of NovaSeq as representative, each run
The sequencing throughput of 60 human genome 30X covering can be reached with output 6TB data within two days；Sequencing throughput and efficiency are compared with Hiseq
Xten has 2~3 times of promotion；With the maturation of high throughput sequencing technologies and the development of clinical research, application, sequencing demands
It can further be promoted.From consumer level physical examination to clinical assistant diagnosis, new challenge can also be brought to sequencing technologies.
Plasma dna also known as recycles cfDNA, is the extracellular dissociative DNA in blood, is about tens to several hundred nucleotide, both
Can exist in the form of DNA- protein complex, be also possible to free DNA fragmentation.Under normal circumstances, plasma dna derives from
The DNA release of a small amount of apoptotic cell.Under health status, the generation and removing of cfDNA is in dynamic balance state, maintains more permanent
Fixed low-level.CfDNA can reflect the situation of human inner cell's metabolism, be the important indicator that health is judged, cfDNA
The change of content and composition and a variety of diseases (including tumour, wound, organ transplant, pregnancy related disorder, infectious diseases, device
Official's failure etc.) it is in close relations.
After the embryo DNA in maternal blood is found, non-invasive diagnosis and detection embryo chromosome multiple sexual abnormality
Hot spot as clinical application research.2008, Lu Yi penetrating judgment, which is awarded, and team takes the lead in detected pregnant woman using high throughput sequencing technologies
Plasma dna counts chromosome proportionate relationship, and micro embryo DNA chromosome number can be detected according to purpose variation and count in blood plasma
Come.With the development of detection technique, technique is included in the forward position of medical diagnosis to develop by world many countries, and is obtained
Great achievement.This inspection is included in residents medical care security system by Countries or city, and China has occupy in this field
World forward position ranks.
The development of sequencing technologies promotes sequencing ability and sequencing efficiency rapidly；The popularization of clinical application, and drive
Sequencing demands generate, to sample library preparation efficiency before being sequenced, more stringent requirements are proposed.Therefore, library preparation efficiency and
Cost will become one of application oriented key factor of high-flux sequence.
The preparation of high-throughput DNA sample, it is therefore an objective to which the both ends DNA to be measured are connected into the connector that upper and microarray dataset matches
DNA to be measured is attached in a sequencing vector by sequence.At present library construction step mainly include nucleic acid extraction (DNA extraction),
End is repaired and 5 ' terminal phosphates, 3 ' ends add the purification process between " A " base, jointing, PCR amplification and step.
This banking process is complicated for operation, higher cost, and the DNA initial amount needed is higher.It is be easy to cause in addition, introducing PCR process
Simultaneously, gene-amplification enzyme performance difference can also introduce certain base preference, cause data in gene Aerosol Pollution between sample
Coverage in group is uneven, leads to the inaccuracy of sequencing result.
Library constructing method optimization be each step reaction system individually react optimization or step unify improved, optimized
It is Main way, such as: DNA is subjected to end reparation and 5 ' terminal phosphate chemical combination simultaneously, 3 ' ends is individually carried out and adds " A " base,
Individually carry out connector connection；Or add " A " base to merge DNA progress end reparation and 5 ' terminal phosphates, 3 ' ends, individually
Carry out connector connection；Or DNA progress end reparation and 5 ' terminal phosphates, 3 ' ends are added into " A " base, jointing three
Step merges；The purpose of optimization, which is for reducing, builds library time and original amounts, and library efficiency is built in raising.And multiple steps are closed
And carried out in a system, reaction efficiency will cause the efficiency one of this reaction step compared with each step individually carries out reaction
Determine degree reduction；And each step is individually reacted, the purification process between step also results in the loss of a part of DNA, and mentions
High operating time cost；Therefore each method is also shortening the reaction time and is guaranteeing to carry out compromise choosing in the contradiction for building library efficiency
It selects.
DNA fragmentation carries out end reparation and 5 ' terminal phosphateizations mainly pass through T4 DNA polymerase, klenow
Fragment, klenow exo- (3 ' -- 5 ' missing), T4 polynucleotide kinase are one of or several, pass through
It adjusts each enzyme dosage and reaction buffer condition optimizes, improve end and repair and 5 ' terminal phosphateizations modification efficiency.DNA piece
3 ' ends of section add " A " base, mainly pass through klenow exo- (3 ' → 5 ') and taq DNA polymerase one or two group
It closes and uses, optimized by adjusting enzyme dosage and reaction buffer condition, improve the efficiency for adding " A " base；DNA connector connects
It connects: mainly being connect by T4 DNA ligase realization with " TA " of vector junctions, buffered by adjusting connection enzyme dosage and reaction
Liquid condition optimizes, and improves the joint efficiency of connector.The library constructing method of existing no PCR amplification, DNA original amounts are flat
In 100ng or so, still there is part sample and be difficult to meet in lower limit also greater than 10ng, the blood plasma dosage of about 1mL, clinical application
The demand of this DNA.
Summary of the invention
The object of the present invention is to provide a kind of construction methods of low initial amount PCR-free high-throughput sequencing library, to make up
Construct that such library takes a long time, is cumbersome, original samples amount is small and can not accurately build the deficiency in library in the prior art.
The construction method of low initial amount PCR-free high-throughput sequencing library provided by the invention the following steps are included:
(1) cfDNA in blood plasma is extracted, end reparation and 5 ' terminal phosphates are carried out to it, purification and recovery end, which is repaired, to be produced
Object；
(2) product is repaired to end after purification, 3 ' ends add " A " base and connector interfaces system to be incorporated into a system
In successively reacted；
(3) connector product is purified, sequencing library is obtained.
CfDNA amount >=1ng in blood plasma described in step (1).
The end of step (1) is repaired and the reaction system of 5 ' terminal phosphates are as follows:
The reaction condition of step (1) are as follows: 20 DEG C of -37 DEG C of 10-30min.
Preferably, the reaction condition of step (1) are as follows: 30 DEG C of 20min.
Step (1) is selected relative to reaction product, is purified with the magnetic bead of 1.5-2.5 times of volume.
In construction method of the invention, in step (2), 3 ' ends add " A " base reaction system are as follows:
10 × reaction buffer 1/10 uses after diluting
DATP 0.2-1mM, 0.5mM are preferred
Klenow exo- (3 ' → 5 ') 0.2-1.0u/ μ L, 0.5u/ μ L is preferred.
In step (2), the reaction system of connector connection are as follows:
2 × connection buffer 1/2 uses after diluting
0.005-0.05 μM of molecular adaptor, 0.01 μM is preferred
T4 DNA ligase 10-50u/ μ L, 20u/ μ L is preferred.
3 ' ends of step (2) add the reaction condition of " A " base to be 20 DEG C~37 DEG C, react 10min~30min；
The reaction condition of connector connection are as follows: 20 DEG C~37 DEG C, react 10min~20min；60 DEG C~75 DEG C, react 10min
~20min；
Preferably, 3 ' ends of step (2) add the reaction condition of " A " base to be 37 DEG C, react 30min；Connector connection
20 DEG C of reaction condition, react 15min；65 DEG C, react 10min.
In the construction method of low initial amount PCR-free high-throughput sequencing library of the invention, the purifying connector of step (3)
Product uses magnetic beads for purifying, and magnetic bead dosage is 0.3-0.8 times of volume of connector product.
The present invention provides application of the construction method in low initial amount DNA high-flux sequence.
Technical solution of the present invention can bring it is following the utility model has the advantages that
(1) sample initial amount requires to reduce.In the prior art, conventional single library construction needs at least 100ng sample,
Since the demand to sample is higher, lead to the rare sample in part because total amount it is less be not achieved build library standard, and library can not be built.
Add " A " base and connector interfaces system to be incorporated into a system the 3 ' end DNA in Library development flow of the present invention and successively completes 3 '
End adds " A " base to connect reaction with connector, omits purification step, has advanced optimized each reaction system, improved efficiency,
Initial amount can be reduced to 1ng, this horizontal DNA initial amount had both reduced the requirement to sample, while also meeting to subsequent
The output and analysis of data, and the reduction of initial amount makes to build the reduction of library reagent cost simultaneously；
(2) during PCR amplification, primer may be partial to certain section of region in conjunction with genome, this section of massive amplification
Region, therefore the covering that the product obtained will appear to genome is uneven, and then sequencing data is made redundancy height occur.This hair
Bright method does not use PCR amplification, reduces the Preference and amplification enzyme that PCR amplification is reduced while the pollution of aerosol
Base preference brought by energy difference, ensure that the validity of data analysis.
(3) connector product purification has adjusted magnetic bead dosage multiple proportions, effectively evades the generation of connector dimer phenomenon.
Detailed description of the invention
Fig. 1-Fig. 3 is respectively the library qPCR solubility curve Quality Control figure of 3 samples constructed in embodiment 1.Carry out library
The detection of qPCR solubility curve will appear a plurality of peak type detection when there are the pcr product of multiple segments；As shown, there was only 1
A detection peak, it was demonstrated that the non-target fragment such as non junction dimer.
Fig. 4-Fig. 6 is respectively G/C content and genome overburden depth in three libraries of 3 samples constructed in embodiment 1
Associated diagram illustrates for different samples column be sequenced to be compared with reference to genome, as unit of 100kb, column will be sequenced and draw
It is divided into several windows, each series of windows G/C content and data cover degree is subjected to correlation statistics；In GC fluctuation range, surveyed
Sequential covering is uniform.
Fig. 7-Fig. 9 is respectively the auspicious qPCR Quality Control figure with the library 1-3 of health kit building of shellfish.
Figure 10-Figure 12 is respectively auspicious " G/C content and the gene overburden depth figure " with health kit building library 1-3 of shellfish.It can
With discovery, the library of this kit building is higher than the high region GC in low GC region overlay depth.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.Biochemical reagents used in following embodiment are equal
It is commercially available.
The construction method (1) of the low initial amount PCR-free high-throughput sequencing library of 1 present invention of embodiment
1, cfDNA is extracted
Maternal blood is acquired using Streck pipe, is transported at room temperature for 24 hours.
First time blood plasma separation, by maternal blood 1600g, 4 DEG C of centrifugation 10min；It checks upper plasma shape, presents saturating
Bright yellowish, the abnormal phenomenon such as no haemolysis are qualification.Upper plasma is drawn into new centrifuge tube, then through 16000g, 4 DEG C of centrifugations
10min；It takes upper plasma to extract for subsequent cfDNA, discards tube bottom retained sediments.Blood plasma cfDNA is carried out using paramagnetic particle method to mention
It takes, the cfDNA after extraction is stored in -80 DEG C of refrigerators.
2, end reparation and phosphorylation reaction
Each reagent reaction dosage see the table below 1:
Repair reaction and phosphorylation reaction in 1 end of table
2.1 PNK buffer, dNTPs and cfDNA are mixed after brief centrifugation, be stored on ice chest；
After 2.2 are mixed T4 archaeal dna polymerase, T4 polynueleotide kinase, klenow segment, it is added to 2.1 mixing
It mixes in object and again.
2.3 reaction conditions: being placed in 30 DEG C, reacts 20min, removes from PCR instrument or metal warm bath instrument, brief centrifugation；
2.4 take 2.3 reaction products, recycle reaction system using 2.0 × Ampure Xp magnetic beads for purifying.
3, the 3 ' end DNA adds " A " to react
Each reagent reaction dosage see the table below:
23 ' end of table adds " A " base
Brief centrifugation after blue buffer, dATP and end reparation product are mixed, is stored on ice chest；It will
Klenow exo- (3 ' → 5 ') is added in mixture even reaction system again；Reaction condition: being placed in 37 DEG C, reacts 30min,
It is removed from PCR instrument or metal warm bath instrument, brief centrifugation.
4, connector connects
Each reagent reaction dosage see the table below:
The connection of 3 connector of table
Brief centrifugation after 2 × ligation buffer, Adaptor and connection product are mixed, is stored in ice chest
On；T4 DNA ligase is added to after mixture and is mixed again；Reaction condition: being placed in 20 DEG C, reacts 15min；65 DEG C, instead
Answer 10min；Connector product carries out purification process according to the purifying magnetic bead that 0.4 × multiple proportions is added in volume ratio (magnetic bead: purified).
5, the library PCR-free prepared by the present invention, is detected through qPCR, and solubility curve and molar concentration meet machine survey
Sequence standard: non junction dimer and molar concentration > 10pM
5.1 survey 3 libraries, and the library using preceding method of the present invention building from 3 different pregnant woman's blood samples is through qPCR
Solubility curve and molar concentration detection, non junction dimer peak and molar concentration meet machine sequencing standard, are shown in Table 4 data.
4 library qPCR quality inspection result of table
| Serial number | Solubility curve detection | Molar concentration (pM) |
| Library 1 | It is unimodal, it is qualified | 166.9 |
| Library 2 | It is unimodal, it is qualified | 164.7 |
| Library 3 | It is unimodal, it is qualified | 139.5 |
5.2 library qPCR solubility curve Quality Control figures are shown in Fig. 1-Fig. 3.
5.3 sequencing datas analyze result
It analyzes coverage condition of each library reads on each chromosome: being examined through tTest, sequencing gained reads is in each dye
Being evenly distributed on colour solid, difference is not significant.G/C content and genome overburden depth associated diagram are shown in Fig. 4-Fig. 6.
5 library reads tTest chromosome of table is distributed significant difference analysis
Column are sequenced and with reference to genome alignment in each sample；Column will be sequenced and with reference to genome with 100kb
Several windows are divided into for unit, every dyeing on sliding statistics sequencing column and reference genome is carried out by unit according to 50kb
Window distribution situation on body carries out T inspection, 3 test sample the data obtaineds and reference genome no significant difference, it was demonstrated that adopt
It can be realized the sequencing library high-throughput to the cfDNA building down to 1ng with banking process of the invention, and institute's measured data is contaminating
Distributing equilibrium on colour solid/genome, data accuracy are high.
The construction method (2) of the low initial amount PCR-free high-throughput sequencing library of 2 present invention of embodiment
Equally the respectively 2ng starting cfDNA from 3 different pregnant woman's blood samples in embodiment 1 is carried out building library, builds library side
Method is same as Example 1, the difference is that:
Step 2.3 reaction condition: being placed in 20 DEG C, reacts 30min.
Step 2.4 recycles reaction system using the Ampure Xp magnetic beads for purifying of 2.5 times of volumes of reaction product.
3 ' ends add in " A " reaction, and reaction condition is 30 DEG C of reaction 30min；
In connector connection reaction, reaction condition is 20 DEG C, reacts 15min；
70 DEG C, react 10min.
Build quality inspection behind library, it is as a result similar to 1 result of embodiment, illustrate 3 test sample the data obtaineds with reference to genome
No significant difference, it was demonstrated that can be realized the sequencing text high-throughput to the cfDNA building down to 1ng using banking process of the invention
Library, and institute's measured data distributing equilibrium on chromosome/genome, data accuracy are high.
Comparative example 1
Using the auspicious foetal chromosome aneuploidy (T13/T18/T21) produced with health Bioisystech Co., Ltd of Beijing shellfish
Detection kit (reversible end termination PCR sequencing PCR), compares with the method for the present invention.The auspicious doctor with health kit of contrast groups shellfish
Treat instrument registration certificate number: state's tool infuses quasi- 20153400461 kits as control experiment, agents useful for same lot number
R1000180901。
1, using Streck pipe extract 3 12 pregnant weeks+maternal blood, be transported at room temperature for 24 hours, check sample without it is broken,
Continue downstream experiment after leakage.
First time blood plasma separation: by maternal blood 1600g, 4 DEG C of centrifugation 10min；
It checks upper plasma shape, transparent yellowish, the abnormal phenomenon such as no haemolysis is presented.
Upper plasma is drawn into new centrifuge tube, then through 16000g, 4 DEG C of centrifugation 10min；300 μ L of upper plasma is taken to mention
CfDNA is taken, for follow-up library building experiment.
Contrast groups: Bei Rui and health kit are tested according to its specification standard operation guidance.
Experimental group of the present invention: carrying out according to 1 laboratory operating procedures of embodiment/process, repairs & phosphorylation → magnetic bead through end
Purifying → 3 ' plus " A " → connector connection → magnetic beads for purifying, the i.e. preparation of completion library.
Molar concentration detection is carried out through qPCR, testing result is shown in Table 6 data.
6 library qPCR quality inspection molar concentration of table
| Project | Bei Rui and health | 1 method of embodiment |
| Sample 1 | 56.6 | 166.9 |
| Sample 2 | 66.4 | 164.7 |
| Sample 3 | 48.9 | 139.5 |
| Mean value | 57.3 | 157.0 |
For the present invention compared with Bei Rui and health library construction Kit, library concentration averagely promotes 2.7 times as the result is shown.Library
Building efficiency greatly promotes.
2, library qPCR solubility curve Quality Control figure is shown in Fig. 1-Fig. 3 (3 library Quality Control figures of embodiment 1), Fig. 7-figure respectively
9 (3 library Quality Control figures of Bei Rui and the building of health kit).
3, sequencing data analyzes result
It analyzes coverage condition of each library reads on each chromosome: being examined through tTest, sequencing gained reads is in each dye
Being evenly distributed on colour solid, difference is not significant.The quality of data is substantially better than Bei Rui and health data.Two methods of 3 sample build library
G/C content and genome overburden depth associated diagram afterwards is shown in Fig. 4-Fig. 6, Figure 10-Figure 12.
7 library reads tTest chromosome of table is distributed significant difference analysis
Column are sequenced and with reference to genome alignment in each sample；Column will be sequenced and with reference to genome with 100kb
Several windows are divided into for unit, every dyeing on sliding statistics sequencing column and reference genome is carried out by unit according to 50kb
Window distribution situation on body carries out T inspection, in the case where level of significance α=0.05, present invention gained test data with
It is compared with reference to genome, hence it is evident that be better than Bei Rui and Kang Fangfa institute measured data.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of construction method of low initial amount PCR-free high-throughput sequencing library, which comprises the following steps:
(1) cfDNA in blood plasma is extracted, end reparation and 5 ' terminal phosphates are carried out to it, product is repaired in purification and recovery end；
(2) product is repaired to end after purification, 3 ' ends add " A " base and connector interfaces system to be incorporated into a system first
After reacted；
(3) connector product is purified, sequencing library is obtained.
2. construction method according to claim 1, which is characterized in that cfDNA amount >=1ng in blood plasma described in step (1).
3. construction method according to claim 1, which is characterized in that the end of step (1) is repaired and 5 ' terminal phosphates
Reaction system are as follows:
4. construction method according to claim 1, which is characterized in that the reaction condition of step (1) are as follows: 20 DEG C of -37 DEG C of 10-
30min。
5. construction method according to claim 1, which is characterized in that step (1) is selected relative to reaction product, and 1.5- is used
The magnetic bead of 2.5 times of volumes is purified.
6. -5 any construction method according to claim 1, which is characterized in that in step (2), 3 ' ends add " A " base
Reaction system are as follows:
10 × reaction buffer 1/10 uses after diluting
dATP 0.2-1mM
Klenow exo-(3’→5’) 0.2-1.0u/μL。
7. construction method according to claim 6, which is characterized in that in step (2), the reaction system of connector connection are as follows:
2 × connection buffer 1/2 uses after diluting
0.005-0.05 μM of molecular adaptor
T4DNA ligase 10-50u/ μ L.
8. -7 any construction method according to claim 1, which is characterized in that 3 ' ends of step (2) add " A " base
Reaction condition is 20 DEG C~37 DEG C, reacts 10min~30min；
The reaction condition of connector connection are as follows: 20 DEG C~37 DEG C, react 10min~20min；60 DEG C~75 DEG C, reaction 10min~
20min。
9. -6 any construction method according to claim 1, which is characterized in that the purifying connector product of step (3) uses
Magnetic beads for purifying, magnetic bead dosage are 0.3-0.8 times of volumes of connector product.
10. application of any construction method of claim 1-8 in low initial amount DNA high-flux sequence.
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| CN112226822A (en) * | 2020-10-26 | 2021-01-15 | 北京百迈客生物科技有限公司 | High-throughput sequencing library construction method for nucleic acid aptamer library |
| CN112899345A (en) * | 2019-12-03 | 2021-06-04 | 暨南大学 | Construction method of ribosome blot sequencing library |
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