CN106801050B - Construction method of circular RNA high-throughput sequencing library and kit thereof - Google Patents

Abstract

The invention discloses a construction method of a circular RNA high-throughput sequencing library and a kit thereof, wherein the construction method sequentially comprises the following steps: (S1) extracting total RNA in the sample; (S2) removing DNA in the sample; (S3) detecting and evaluating the quality of the total RNA in the sample, and determining that the quality of the RNA meets the requirement; (S4) removing rRNA; (S5) removing the linear RNA; (S6) constructing a circular RNA library for high-throughput sequencing. The construction method of the circular RNA high-throughput sequencing library is efficient and stable, the residual ratio of rRNA is low, the detection efficiency of the circular RNA is high, the data repeatability is good, the success rate of sequencing result verification is high, and the method is particularly suitable for samples with poor RNA quality such as FFPE tissues.

Description

Construction method of circular RNA high-throughput sequencing library and kit thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a construction method of a circular RNA high-throughput sequencing library and a kit thereof.

Background

Circular RNA (circRNA) is a new member of the RNA family that is distinguished from traditional linear RNA by being a non-coding RNA molecule that does not have a 5 'terminal cap and a 3' terminal poly (a) tail and is covalently bonded to form a circular structure. Circular RNA has been discovered as early as 1980, but for a long time, due to the limitations of the state of the art, circular RNA is considered as a by-product of the erroneous variable cleavage, a very rare phenomenon in nature, and even as a result of genetic accidents or experimental artifacts, has not received academic attention. With the development of deep RNA sequencing and large-scale bioinformatics techniques, researchers have only discovered that a large number of circular RNA molecules exist in organisms. Salzman et al discovered in 2012 that circular RNA is widely present in the human transcriptome by high throughput sequencing, and thus, have attracted interest in circular RNA research. In 2013, Hansen et al find that CDR1as/CiRS-7 can be used as miR-7 sponge to regulate gene expression, and prove that the important function of circular RNA in a human body is achieved. Recent research shows that the circular RNA has a closed circular structure, is mainly generated through atypical variable shearing processing, is widely present in various biological cells, has the characteristics of stable structure, difficult degradation by RNA enzyme, high expression abundance, good conservation among species, tissue and space specificity and the like. At present, research shows that the circular RNA is related to diseases such as neurodevelopment, atherosclerosis, myotonic dystrophy, cancer and the like, and the detection of the existence of the circular RNA in human saliva and blood indicates that the circular RNA can stably exist in clinical specimens such as blood, urine, ascites and the like, and the characteristics enable the circular RNA to have wide prospects in the development and application of novel disease diagnosis and treatment methods.

CN 104388548A discloses a method for high throughput circular RNA sequencing, which comprises: artificially synthesizing exogenous linear ERCC-0004-RNA and exogenous circular ERCC-0013-RNA; performing quality control on the exogenous circular ERCC-0013-RNA; mixing exogenous linear ERCC-0004-RNA and exogenous annular ERCC-0013-RNA according to an equimolar ratio to obtain mixed exogenous RNA; adding mixed exogenous RNA into total RNA of a sample to be sequenced to obtain a first mixture; removing ribosomal RNA from the first mixture to obtain a second mixture; labeling the second mixture with biotin at the 3' end, and removing lasso RNA and linear RNA labeled with biotin to obtain a third mixture; removing the linear RNA in the third mixture to obtain a fourth mixture; standard high throughput transcriptome sequencing was performed on the fourth mixture and biological analysis was performed on the sequencing data. The method is complicated in steps and requires the prior synthesis of exogenous RNA.

As circular RNA can not be directly separated, a standard method for constructing a circular RNA library does not exist in the literature so far, most researches adopt a method of removing rRNA and linear RNA, and then constructing a library for high-throughput sequencing, a small part of researches also adopt a method of directly constructing a library after removing rRNA, or directly constructing a library after removing linear RNA, and a very small part of researches adopt a method of constructing a library after removing PolyA + RNA. The methods have the defects of incomplete rRNA removal, poor data repeatability and the like. Therefore, how to efficiently and stably construct a library for circular RNA high-throughput sequencing is still a technical problem to be solved.

In addition, no special circular RNA library construction kit exists in the market at present, and scientific researchers need to purchase products such as RNA extraction, rRNA removal, linear RNA removal and library construction from multiple companies such as QIAGEN, Illumina, Backman, NEB and Life technology respectively and then search for proper conditions to construct libraries. On one hand, the multiple reagent combination library is expensive, on the other hand, scientific researchers with higher experimental skills spend longer time to search for the optimal experimental conditions, and the progress of circular RNA research is severely limited, so that a special kit which can efficiently and stably construct a high-throughput sequencing library for circular RNA is needed to be developed.

Disclosure of Invention

In view of the above problems, the present inventors have repeatedly studied and analyzed circular RNA sequencing to develop a method capable of efficiently and stably constructing a circular RNA high-throughput sequencing library, which has a low rRNA residual ratio and good data reproducibility, and which is particularly suitable for samples with poor RNA quality such as FFPE tissue.

In order to achieve the above object, the present invention provides a method for constructing a circular RNA high-throughput sequencing library, which sequentially comprises the following steps:

(S1) extracting total RNA in the sample;

(S2) removing DNA in the sample;

(S3) detecting and evaluating the quality of the total RNA in the sample, and determining that the quality of the RNA meets the requirement;

(S4) removing rRNA;

(S5) removing the linear RNA;

(S6) constructing a circular RNA library for high-throughput sequencing.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S1) of extracting total RNA in the sample is performed by using a Trizol method, which is particularly suitable for tissue and cell samples.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S2) of removing DNA in the sample is performed by DNase I digestion.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S3) of detecting the total RNA mass in the sample sequentially comprises the following steps:

(S31) detecting the degree of RNA degradation and presence or absence of contamination using 1% agarose gel electrophoresis;

(S32) detecting the purity of the RNA, namely the OD260/280 ratio, by using an ultraviolet spectrophotometer;

(S33) the integrity of the RNA is tested.

More preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S33) of detecting the integrity of RNA is performed by using Agilent 2100 bioanalyzer.

In the present invention, OD (optical density) represents the optical density absorbed by the substance to be detected, and the OD260/280 ratio is the ratio of absorptivities at wavelengths of 260nm and 280nm, and is used for judging the purity of RNA. Theoretically, the OD260/280 ratio in the case of pure RNA is 2, where OD260 represents the absorbance of nucleic acids and OD280 represents the absorbance of proteins.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the rRNA removal in the step (S4) is performed by RNase H digestion.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S4) of removing rRNA sequentially comprises the following steps:

(S41) mixing DNA probes complementary to the rRNA sequences in equal mass to form a DNA probe library;

(S42) mixing and hybridizing the obtained DNA probe library with RNA which is detected and evaluated to determine the quality of the RNA according with the requirement, and forming a DNA-RNA hybrid;

(S43) digesting the obtained DNA-RNA hybrid with RNase H;

(S44) digesting the DNA probe with DNase I to obtain rRNA-depleted RNA;

(S45) the concentration of rRNA-depleted RNA was determined by fluorometry.

Preferably, in the method for constructing a circular RNA high-throughput sequencing library, the weight ratio of the DNA probe pool to RNA used in the step (S42) is (1-2): 1, preferably 1: 1.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the hybridization in the step (S42) is performed under the condition of hybridizing at a temperature of 95 ℃ for 2 minutes, and then the temperature is slowly decreased to 45 ℃ at a rate of 0.1 ℃/S.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the DNA-RNA hybrid obtained by RNaseH digestion in the step (S43) is digested at 37 ℃ for 30 minutes.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the fluorometer used in the step (S45) of determining the concentration of rRNA-depleted RNA is performed by a Qubit fluorometer.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the linear RNA removal in the step (S5) is performed by RNase R digestion, wherein the dosage ratio of RNase R to RNA is (2.5-3.5) U:1ug, preferably 3:1, that is, 3 active units (U) of RNase R are added per 1 microgram of RNA.

More preferably, in the method for constructing the circular RNA high-throughput sequencing library, the RNase R digestion method used in the step (S5) of removing linear RNA is performed under digestion conditions of 35-45 deg.C, preferably 37 deg.C, for 20-40 minutes, preferably 30 minutes.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S6) of constructing the circular RNA library for high-throughput sequencing is performed by a dUTP method.

Preferably, in the method for constructing a circular RNA high-throughput sequencing library, the step (S6) of constructing a circular RNA library for high-throughput sequencing sequentially comprises the following steps:

(S61) RNA fragmentation;

(S62) first strand synthesis: the method is carried out by adopting a method of reverse transcriptase and random primers;

(S63) second Strand Synthesis: digesting an RNA strand in the RNA/cDNA hybrid by RNase H, and then synthesizing a second strand by DNA polymerase I;

(S64) end repair: using End Repair Mix to form blunt-ended dsDNA and adding phosphate group at 5' End;

(S65) adding A tail: add A tail to dsDNA ends using Klenow 3'-5' exo-;

(S66) Y-type Adapter connection: t4 DNA Ligase is used for connecting the Adapter and the dsDNA with an A tail added;

(S67) second strand digestion and library amplification: digesting the second strand with UNG enzyme;

(S68) quality testing and preparation of the indexing library.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the RNA fragmentation condition in the step (S61) is fragmentation at a temperature of 90-96 ℃, preferably 94 ℃ for 3-6 minutes, preferably 5 minutes.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the peak after RNA fragmentation in the step (S61) is located at 300-350 bp.

Preferably, in the method for constructing the circular RNA high throughput sequencing library, the step (S62) of first strand synthesis inhibits DNA-dependent DNA polymerase activity of reverse transcriptase using actinomycin D to retain directional information of strands.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S66) of Y-type Adapter ligation uses phosphorothioate linkage at the end of the Adapter to link T, so as to prevent formation of Adapter dimer, and further preferably, after purification of the ligation product once using 1 xbeads, 0.7 x/0.2 xbeads is used for fragment size screening to select fragments with appropriate size.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S68) of quality inspection and preparation of the indexing library sequentially comprises the following steps:

(S681) detecting a library concentration;

(S682) detecting the quality of the library by using Agilent 2100;

(S683) different indexed libraries are mixed according to the library concentration, ready for on-machine sequencing.

Compared with the conventional method, the construction method of the circular RNA high-throughput sequencing library provided by the invention has the advantages of high efficiency, stability, low rRNA residual proportion, high circular RNA detection efficiency, good data repeatability and high sequencing result verification success rate, and is particularly suitable for samples with poor RNA quality such as FFPE tissues and the like.

In addition, the invention also provides a kit for constructing the circular RNA high-throughput sequencing library, which comprises:

(1) cyclic RNA enrichment reagent, DNase I, RNase H and RNaseR;

(2) species-specific DNA probes;

(3) various enzymes required for library construction: DNA Polymerase I, Large (Klenow) Fragment, T4Polynucleotide Kinase (T4 PNK), Klenow Fragment, Escherichia coli UDG, T4 DNALigase (Rapid), Phusion High-Fidelity DNA Polymerase;

(4) magnetic beads, dNTP, nucleic Free Water, dUTP, a joint, a primer and the like,

wherein the species-specific DNA probe is complementary to an rRNA sequence of the corresponding species.

The kit for constructing the circular RNA high-throughput sequencing library is simple to operate, optimized in conditions and low in cost, and is a powerful tool for circular RNA research.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the method for constructing the circular RNA high-throughput sequencing library establishes a species-specific DNA probe library, adopts RNaseH to remove rRNA combined with the probe, and adopts RNaseR to further enrich the circular RNA, so the rRNA residue proportion is low, the circular RNA detection efficiency is high, the data repeatability is good, the sequencing result verification success rate is high, and the method is particularly suitable for samples with poor RNA quality such as FFPE tissues and the like;

(2) the kit for constructing the circular RNA high-throughput sequencing library is simple to operate, optimized in conditions and low in cost, and is a powerful tool for circular RNA research.

Drawings

FIG. 1 is a flow chart of one embodiment of the method for constructing a circular RNA high throughput sequencing library according to the present invention;

FIG. 2-1 shows the result of quality control of a circular RNA high throughput sequencing library by Agilent 2100 after completion of library construction in a cell sample according to example 1 of the present invention;

FIG. 2-2 shows the result of quality control of a circular RNA high throughput sequencing library by Agilent 2100 after completion of library construction in a fresh liver cancer tissue sample according to example 2 of the present invention;

FIGS. 2-3 show the results of quality control of the circular RNA high throughput sequencing library by Agilent 2100 after completion of library construction in FFPE sample according to example 3 of the present invention;

FIG. 3 shows the QPCR verification results of 20 circular RNAs selected after the completion of the sequencing of the cell samples in example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Specifically, as shown in fig. 1, an embodiment of the present invention provides a method for constructing a circular RNA high-throughput sequencing library, which sequentially includes the following steps:

s1, extracting total RNA in the sample;

the extraction of total RNA from a sample is carried out using Trizol, which is particularly suitable for tissue and cell samples.

S2, removing the DNA in the sample.

Preferably, the removal of DNA from the sample is performed by DNase I digestion.

And S3, detecting and evaluating the total RNA quality in the sample, and determining that the RNA quality meets the requirements.

Preferably, detecting and assessing the quality of total RNA in a sample comprises the following steps in sequence:

(S31) detecting the degree of RNA degradation and presence or absence of contamination using 1% agarose gel electrophoresis;

(S32) detecting the purity of the RNA, namely the OD260/280 ratio, by using an ultraviolet spectrophotometer;

(S33) the integrity of the RNA is tested.

The step (S33) of detecting the integrity of the RNA was performed using an Agilent 2100 bioanalyzer.

S4, removing rRNA.

The rRNA removal was performed by RNase H digestion.

The rRNA removal by RNase H digestion sequentially comprises the following steps:

(S41) mixing DNA probes complementary to the rRNA sequences in equal mass to form a DNA probe library;

(S42) mixing and hybridizing the obtained DNA probe library with RNA which is detected and evaluated to determine the total RNA quality in the sample and meets the requirement of the RNA quality to form a DNA-RNA hybrid;

(S43) digesting the obtained DNA-RNA hybrid with RNase H;

(S44) digesting the DNA probe with DNase I to obtain rRNA-depleted RNA;

(S45) the concentration of rRNA-depleted RNA was determined by fluorometry.

The mass ratio of the DNA probe library to the RNA used in the step (S42) is (1-2): 1, preferably 1: 1.

The hybridization in the step (S42) is carried out under the conditions of hybridization at a temperature of 95 ℃ for 2 minutes, followed by slow temperature reduction at a rate of 0.1 ℃/S to 45 ℃ and incubation at 45 ℃ for 5 minutes.

The conditions used for digesting the obtained DNA-RNA hybrid with RNase H in the step (S43) are digestion at a temperature of 37 ℃ for 30 minutes.

The fluorometer used in the step (S45) of determining the concentration of rRNA-depleted RNA was performed by a Qubit fluorometer.

The product obtained in said step (S45) is further detected by an Agilent 2100 instrument to confirm that a typical peak pattern of 18S, 28S rRNA can not be seen.

S5, linear RNA was removed.

Linear RNA removal was performed by RNase R digestion with an amount ratio of RNase R to RNA of (2.5-3.5) U:1ug, preferably 3:1, i.e.3 units (U) of RNase R per 1. mu.g of RNA are added.

The digestion conditions of the RNase R digestion method for removing linear RNA are digestion at 35-45 deg.C, preferably 37 deg.C for 20-40 min, preferably 30 min.

S6, constructing a circular RNA library for high-throughput sequencing.

Construction of circular RNA libraries for high throughput sequencing was performed using the dUTP method.

The dUTP method for constructing the circular RNA library for high-throughput sequencing sequentially comprises the following steps:

(S61) RNA fragmentation;

(S62) first strand synthesis: the method is carried out by adopting a method of reverse transcriptase and random primers, and the raw materials are dNTPs (25 mM of each of dA, dC, dG and dT);

(S63) second Strand Synthesis: the RNA strand in the RNA/cDNA hybrid was digested with RNase H, followed by synthesis of the second strand by DNA polymerase I starting with dUTP mix (10mM dA, dC, dG and 20mM dU);

(S64) end repair: using End Repair Mix to form blunt-ended dsDNA and adding phosphate group at 5' End;

(S65) adding A tail: add A tail to dsDNA ends using Klenow 3'-5' exo-;

(S66) Y-type Adapter connection: t4 DNA Ligase is used for connecting the Adapter and the dsDNA with an A tail added;

(S67) second strand digestion and library amplification: digesting the second strand with UNG enzyme;

(S68) quality testing and preparation of the indexing library.

The condition for fragmenting RNA in said step (S61) is fragmentation at a temperature of 90-96 deg.C, preferably 94 deg.C, for 3-6 minutes, preferably 5 minutes.

The peak after RNA fragmentation in the step (S61) is located at 300-350 bp.

The step (S62) first strand synthesis inhibits DNA-dependent DNA polymerase activity of reverse transcriptase using actinomycin D to retain strand directionality information.

In the step (S66), the T-terminal of the Adapter in the Y-type Adapter ligation is linked by phosphorothioate bond to prevent formation of Adapter dimer, and further preferably, after the ligation product is purified once by 1 × beads, the fragments are screened by 0.7 × 0.2 × beads to select the appropriate size of the fragments.

The step (S68) of quality testing and preparing an indexing library comprises the steps of:

(S681) detecting the library concentration by a Qubit fluorometer;

(S682) detecting the quality of the library by using Agilent 2100;

(S683) different indexed libraries are mixed according to the library concentration, ready for on-machine sequencing.

Compared with the conventional method, the construction method of the circular RNA high-throughput sequencing library provided by the invention has the advantages of high efficiency, stability, low rRNA residual proportion, high circular RNA detection efficiency, good data repeatability and high sequencing result verification success rate, and is particularly suitable for samples with poor RNA quality such as FFPE tissues and the like.

In order to make the objects and advantages of the invention more concise, the invention will be described in more detail with reference to the following examples, to which it is in no way limited. The following examples are merely preferred examples of the present invention and are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Example 1 construction of circular RNA high-throughput sequencing library in cell samples

(S1) extraction of Total RNA from cell samples

Taking cell sample 1X 10⁷After washing once with PBS at 4 ℃, 1mL of trizol was added to each well of the 6-well plate, and the plate was repeatedly blown 10 times with a 1mL pipette tip. The sample was collected in an EP tube, 200. mu.L of chloroform was added thereto, and the mixture was mixed by turning upside down for 30 seconds and allowed to stand for 3 min. The lysate is then centrifuged for 15min at a temperature of 4 ℃ and a speed of 12000rpm, and the solution is divided into three layers, the upper layer being RNA dissolved in the aqueous phase. Collecting the upper layer solution, adding equal volume of isopropanol, mixing, and standing at 4 deg.C for 10 min. After which it was again centrifuged at a temperature of 4 ℃ and a rotational speed of 12000rpm for 10min and the supernatant removed. Then 1mL 75% ethanol was added to the pellet and the pellet was resuspended by mixing the pellet upside down. The pellet was then also centrifuged at a temperature of 4 ℃ and a rotational speed of 12000rpm for 10min and the supernatant removed. The precipitate was then dried at room temperature for 15min until the tube wall was free of liquid. Followed by the addition of 25. mu.L DEPC H₂O dissolves RNA to obtain an RNA solution.

(S2) removing DNA from the cell sample

An experimental system was prepared as follows:

the above experimental system was then incubated at room temperature for 30min, after which 300. mu.L of Buffer RP was added and vortexed for 15s, followed by standing for 10min to inactivate DNase I. Thereafter 250. mu.L of absolute ethanol were added and vortexed for 15s, followed by brief centrifugation to remove droplets on the tube wall. The RNA was then purified using a Hipure RNA column and 25. mu.L DEPC H₂And O, eluting to obtain the RNA sample after DNA removal.

(S3) detecting and evaluating the total RNA quality in the sample, and determining that the RNA quality meets the requirements

And detecting the RNA degradation degree in the RNA sample after the DNA is removed by using 1% agarose gel electrophoresis, and determining whether the sample is polluted, wherein the result shows that the sample is pollution-free and the RNA degradation degree is small. Then, the purity of the RNA is detected by using an ultraviolet spectrophotometer, wherein the OD260/280 ratio is 1.95, and the purity is higher. RNA integrity was then analyzed by an Agilent 2100 bioanalyzer, which showed good RNA integrity and a RIN of 9.2.

(S4) removal of rRNA

195 DNA probes of 50bp, which are complementary to rRNA sequences in cells, are designed and mixed together in equal mass to form a DNA probe library, and then an experimental system shown as follows is prepared:

DNA Probe library 5. mu.g

DNA-depleted RNA sample 5. mu.g

5×hybridization buffer 5μL

(the composition was 1M NaCl, 0.5M Tris-HCl, pH 7.4);

DEPC water was added to 25. mu.L.

The above test system was mixed at 95 ℃ for two minutes, and then the temperature of the test system was lowered to 45 ℃ at a rate of 0.1 ℃/s to form a DNA-RNA hybrid. Then 10. mu.L of RNase H (5U/. mu.L) and 5. mu.L of 10 XRNase H Reaction Buffer (composition: 500mM Tris-HCl, 750mM KCL, 30mM MgCl) preheated to 37 ℃ were added₂100mm dtt, pH 8.3) and 5 μ L DEPC water were added to the DNA-RNA hybrids. RNA hybridized to DNA was then digested at 37 ℃ for 30min and purified using 2.2 × RNA Clean XPbeads. Thereafter, 5. mu.L of TurboDNase I (2U/. mu.L) was added to the reaction system and the DNA probe was digested at 37 ℃ for 30 min. Subsequently, rRNA-depleted RNA was obtained by purification using 2.2 XRNA Clean XP beads, and the concentration of rRNA-depleted RNA was measured by a Qubit fluorometer, showing a concentration of 21.78 ng/. mu.L.

(S5) removal of Linear RNA

An experimental system was prepared as follows:

the above system was digested in a 37 ℃ water bath for 30min to obtain linear RNA-depleted RNA, and the concentration thereof was measured with a Qubit fluorometer, showing a concentration of 4.60 ng/. mu.L.

(S6) construction of circular RNA libraries for high throughput sequencing

(S61) fragmentation of RNA

An experimental system was prepared as follows:

5×First Strand Buffer 8μL

linear RNA-depleted RNA 10. mu.L

Nuclease free H₂O 2μL；

And (3) putting the experimental system into a PCR instrument for incubation, wherein the procedure of the PCR instrument is as follows: after 5min at 94 ℃, the RNA was immediately placed on ice to obtain fragmented RNA.

(S62) first strand synthesis:

an experimental system was prepared as follows:

and (3) putting the experimental system into a PCR instrument for incubation, wherein the procedure of the PCR instrument is as follows: 3min at 65 ℃ then placed on ice and 4. mu.L of nucleic free H was added₂O, 1. mu.L of 100mM DTT, 0.1. mu.L of 25mM dNTPs, 0.5. mu.L of SupeRase-In, 0.5. mu. L M-MulVReverse Transcriptase, and 4. mu.g of Actinomycin D. The reaction was then incubated in a PCR instrument programmed to: 10min at 25 ℃; 50min at 42 ℃; 15min at 70 ℃; hold at 4 ℃. Then 38. mu.L RNA Clean XP and 19. mu.L 100% ethanol was added for purification and 16. mu.L nucleic Free H was used₂O elution to obtain RNA/cDNA hybrids, and transfer to a new tube.

(S63) second Strand Synthesis

An experimental system was prepared at 0 ℃ as follows:

the above experimental system was incubated at 16 ℃ for 2.5 h. Subsequently 38. mu.L of RNA Clean XP and 19. mu.L of ethanol were added for purification and eluted with 32. mu.L of Qiagen EB to give dsDNA.

(S64) end repair

An experimental system was prepared at 0 ℃ as follows:

the above experimental system was placed in a PCR instrument and maintained at 20 ℃ for 30 min. Followed by addition of 28. mu.L of RNA CleanXP and 14. mu.L of ethanol and application of 17. mu.L of nucleic Free H₂And eluting to obtain the dsDNA after the end is repaired.

(S65) adding A tail

An experimental system was prepared as follows:

then incubated at 37 ℃ for 30 min. Followed by 28. mu.L of AMPure XP beads and 14. mu.L of ethanol and 10. mu.L of nucleic Free H₂O elution to obtain dsDNA with an A tail added.

(S66) Y-type Adapter connection:

an experimental system was prepared at 0 ℃ as follows:

the above experimental system was placed in a PCR instrument and incubated at 20 ℃ for 20min, then mixed with 24. mu.L of "12P XP" beads and incubated at room temperature for 6min, after which the supernatant was retained and mixed with 12. mu.L of AMPure XP beads and 5. mu.L of 40 wt% PEG8000, and incubated at room temperature for 6min, then with 10. mu.L of nucleic Free H₂Eluting twice with O to obtain eluent, and mixing with waterThe eluate was mixed with 12 μ L AMPure XP, incubated again at room temperature for 6min, and then eluted 1 time with 30 μ L qiagen eb to give the product.

(S67) second Strand digestion and library amplification

mu.L of the above product was mixed with 1. mu.L of Uracil DNA Glycosylase and incubated at 37 ℃ for 30min to give UNG digested DNA. The experimental system shown below was then formulated at 0 ℃:

performing near-PCR amplification on the experimental system under the following cycle conditions: 30s at 94 ℃; (98 ℃ for 10 s; 65 ℃ for 30 s; 72 ℃ for 30s) for 15 cycles; 5min at 72 ℃; the temperature of the experimental system was then maintained at 4 ℃. Subsequently, 43. mu.L of AMPure XPbeads were added for purification and eluted using 12. mu.L of Qiagen EB to generate a library.

(S68) quality control and preparation of an indexed library

The concentration of the library was first determined using a Qubit fluorometer, resulting in 4.06 ng/. mu.L. Then, the Agilent 2100 bioanalyzer is used for detecting the quality of the library, the detection result is shown in FIG. 2, and the result shows that the size of the insert is qualified, the peak type is single, and the peak value is about 200-500 bp. Different indexing libraries are then mixed according to the detected concentration. And (4) detecting the concentration of the library by using a Qubit fluorometer, and then starting to continue the machine sequencing work.

Example 2 construction of circular RNA high throughput sequencing library in fresh liver cancer tissue samples

(S1) extraction of Total RNA from fresh liver cancer tissue samples

A fresh liver cancer tissue sample (50 g) was taken, washed once with PBS at 4 ℃ and the tissue was minced. 1ml of trizol was then added and homogenized 15 times with a portable high-speed disperser. Thereafter, the sample was collected in an EP tube, 200. mu.L of chloroform was added thereto, and the mixture was mixed by turning upside down for 30 seconds and then allowed to stand for 3 min. The lysate is then centrifuged for 15min at a temperature of 4 ℃ and a speed of 12000rpm, and the solution is divided into three layers, the upper layer being RNA dissolved in the aqueous phase. And taking the upper solution, adding isopropanol with the same volume into the upper solution, uniformly mixing the solution and standing the mixture for 10 min. Thereafter again at a temperature of 4 ℃ and a rotation of 12000rpmCentrifuge rapidly for 10min and remove supernatant. Then 1mL 75% ethanol was added to the pellet and the pellet was resuspended by mixing the pellet upside down. The pellet was then also centrifuged at a temperature of 4 ℃ and a rotational speed of 12000rpm for 10min and the supernatant removed. The precipitate was then dried at room temperature for 15min until the tube wall was free of liquid. Then 30. mu.L DEPC H was added₂O dissolves RNA to obtain an RNA solution.

(S2) removing DNA from the fresh tissue sample

An experimental system was prepared as follows:

the above experimental system was then incubated at room temperature for 30min, after which 300. mu.L of Buffer RP was added and vortexed for 15s, followed by standing for 10min to inactivate DNase I. Thereafter 250. mu.L of absolute ethanol was added and vortexed for 15s, followed by centrifugation to remove droplets on the tube wall. The RNA was then purified using a Hipure RNA column and 25. mu.L DEPC H₂And O, eluting to obtain the RNA sample after DNA removal.

(S3) detecting and evaluating the total RNA quality in the sample, and determining that the RNA quality meets the requirements

And detecting the RNA degradation degree in the RNA sample after the DNA is removed by using 1% agarose gel electrophoresis, and determining whether the sample is polluted, wherein the result shows that the sample is pollution-free and the RNA degradation degree is small. Then, the purity of the RNA is detected by using an ultraviolet spectrophotometer, wherein the OD260/280 ratio is 2.05, and the purity is higher. RNA integrity was then analyzed by an Agilent 2100 bioanalyzer, which showed good RNA integrity with an RIN of 7.1.

(S4) removal of rRNA

195 DNA probes of 50bp which are complementary with rRNA sequences in fresh tissues are designed and mixed together in equal mass to form a DNA probe library, and then an experimental system shown as the following is prepared:

DNA Probe library 5. mu.g

DNA-depleted RNA sample 5. mu.g

5×hybridization buffer 5μL

(the component is 1M NaCl, 0.5M Tris-HCl, pH7.4)

DEPC water was added to 25. mu.L.

The above test system was mixed at 95 ℃ for two minutes, and then the temperature of the test system was lowered to 45 ℃ at a rate of 0.1 ℃/s to form a DNA-RNA hybrid. Then 10. mu.L of RNase H (5U/. mu.L) and 5. mu.L of 10 XRNase H Reaction Buffer (composition: 500mM Tris-HCl, 750mM KCL, 30mM MgCl) preheated to 37 ℃ were added₂100mm dtt, pH 8.3) and 5 μ L DEPC water were added to the DNA-RNA hybrids. The RNA hybridized to the DNA was then digested at 37 ℃ for 30min and purified using 2.2 × RNA Clean XP beads. Then, 5. mu.L of LTurbo DNase I (2U/. mu.L) was added to the reaction system and the DNA probe was digested at 37 ℃ for 30 min. Subsequent purification using 2.2 × RNA Clean XPbeads gave rRNA depleted RNA, while the concentration of rRNA depleted was determined using a Qubit fluorometer, which showed a concentration of 24.8 ng/. mu.L.

(S5) removal of Linear RNA

An experimental system was prepared as follows:

the above system was digested in a 37 ℃ water bath for 30min to obtain linear RNA-depleted RNA, and the concentration thereof was measured with a Qubit fluorometer, showing a concentration of 8.2 ng/. mu.L.

(S6) construction of circular RNA libraries for high throughput sequencing

(S61) fragmentation of RNA

An experimental system was prepared as follows:

5×First Strand Buffer 8μL

linear RNA-depleted RNA 10. mu.L

Nuclease free H₂O 2μL；

And (3) putting the experimental system into a PCR instrument for incubation, wherein the procedure of the PCR instrument is as follows: after 5min at 94 ℃, the RNA was immediately placed on ice to obtain fragmented RNA.

(S62) first strand synthesis:

an experimental system was prepared as follows:

and (3) putting the experimental system into a PCR instrument for incubation, wherein the procedure of the PCR instrument is as follows: 3min at 65 ℃ then placed on ice and 4. mu.L of nucleic free H was added₂O, 1. mu.L of 100mM DTT, 0.1. mu.L of 25mM dNTPs, 0.5. mu.L of SupeRase-In, 0.5. mu. L M-MulVReverse Transcriptase, and 4. mu.g of Actinomycin D. The reaction was then incubated in a PCR instrument programmed to: 10min at 25 ℃; 50min at-42 ℃; 15min at-70 ℃; hold at-4 ℃. Then 38. mu.L of RNA Clean XP and 19. mu.L of 100% ethanol were added for purification and 16. mu.L of nucleic Free H was used₂O elution to obtain RNA/cDNA hybrids, and transfer to a new tube.

(S63) second Strand Synthesis

An experimental system was prepared at 0 ℃ as follows:

the above experimental system was incubated at 16 ℃ for 2.5 h. Subsequently 38. mu.L of RNA Clean XP and 19. mu.L of ethanol were added for purification and eluted with 32. mu.L of Qiagen EB to give dsDNA.

(S64) end repair

An experimental system was prepared at 0 ℃ as follows:

the above experimental system was placed in a PCR instrument and maintained at 20 ℃ for 30 min. Followed by addition of 28. mu.L of RNA CleanXP and 14. mu.L of ethanol and application of 17. mu.L of nucleic Free H₂And eluting to obtain the dsDNA after the end is repaired.

(S65) adding A tail

An experimental system was prepared as follows:

then incubated at 37 ℃ for 30 min. Followed by 28. mu.L of AMPure XP beads and 14. mu.L of ethanol and 10. mu.L of nucleic Free H₂O elution to obtain dsDNA with an A tail added.

(S66) Y-type Adapter connection:

an experimental system was prepared at 0 ℃ as follows:

the above experimental system was placed in a PCR instrument and incubated at 20 ℃ for 20min, then mixed with 24. mu.L of "12P XP" beads and incubated at room temperature for 6min, after which the supernatant was retained and mixed with 12. mu.L of AMPure XP beads and 5. mu.L of 40 wt% PEG8000, and incubated at room temperature for 6min, then with 10. mu.L of nucleic Free H₂O elution twice to give an eluent, after which the eluent was mixed with 12 μ L AMPure XP, incubated again at room temperature for 6min, and then eluted 1 time using 30 μ L qiagen eb to give the product.

(S67) second Strand digestion and library amplification

mu.L of the above product was mixed with 1. mu.L of Uracil DNA Glycosylase and incubated at 37 ℃ for 30min to give UNG digested DNA. The experimental system shown below was then formulated at 0 ℃:

performing near-PCR amplification on the experimental system under the following cycle conditions: 30s at 94 ℃; circulating for 15 cycles (-98 deg.C, 10s (-65 deg.C, 30s (-72 deg.C, 30 s)); 5min at-72 ℃; the temperature of the experimental system was then maintained at-4 ℃. Subsequently, 43. mu.L of AMPureXP beads were added for purification and eluted using 12. mu.L of Qiagen EB to generate a library.

(S68) quality control and preparation of an indexed library

The concentration of the library was first measured using a Qubit fluorometer and found to be 5.16. Then, the Agilent 2100 bioanalyzer is used for detecting the quality of the library, the detection result is shown in figure 2, the size of the insert is qualified, the peak type is single, and the peak value is about 200-500 bp. Different indexing libraries are then mixed according to the detected concentration. And (4) detecting the concentration of the library by using a Qubit fluorometer and then starting the sequencing work on the computer.

Example 3 construction of circular RNA high throughput sequencing libraries in FFPE samples

(S1) extraction of Total RNA from FFPE samples

The FFPE samples were sliced into 8 μm thick pieces and immediately transferred to a 1.5mL centrifuge tube for 7 pieces. Then 1mL of xylene was vortexed vigorously for 30s and centrifuged at 14000rpm for 2 min. Then 1mL ethanol was added to the sample and vortexed for 30s, centrifuged again for 2min at 14000rpm, the supernatant removed and the pellet retained. Drying at 37 deg.C for 15min to remove ethanol. Subsequently, 200. mu.L of lysis buffer and 20. mu.l of protease K were added to the pellet and vortexed. Thereafter a water bath was carried out at a temperature of 55 ℃ for 15min, and thereafter at a temperature of 80 ℃ for 15 min. After brief centrifugation at low speed, 200. mu.L of buffer was added to the sample and vortexed for 20 s. 600 μ L of absolute ethanol was then added to the sample and vortexed for 20 s. The mixture was then transferred to an adsorption column and centrifuged at 8000rpm for 50s, the effluent was decanted and the column was mounted in a collection tube. Subsequently 500. mu.L of the rinse solution was added to the column, centrifuged at 8000rpm for 50s, the effluent was decanted, and the column was mounted in a collection tube. Thereafter, 500. mu.L of the rinsing solution was added to the column, and the column was centrifuged at 8000rpm for 50 seconds. The effluent was decanted and the column was mounted in a collection tube. The column was then spun dry by centrifugation at 13000rpm for 3min and transferred to a new 1.5mL centrifuge tube. Then, 30. mu.L of DEPC water was added to the center of the membrane of the column and left standing for 2min, followed by centrifugation at 13000rpm for 1min to obtain an RNA solution.

(S2) removing DNA in the FFPE sample

An experimental system was prepared as follows:

then the above-mentioned experimental body is mixedThe line was incubated at room temperature for 30min, after which 300. mu.L Buffer RP was added and vortexed for 15s, followed by standing for 10min to inactivate DNase I. Thereafter 250. mu.L of absolute ethanol was added and vortexed for 15s, followed by centrifugation to remove droplets on the tube wall. The RNA was then purified using a Hipure RNA column and 25. mu.L DEPC H₂And O, eluting to obtain the RNA sample after DNA removal.

(S3) detecting and evaluating the total RNA quality in the sample, and determining that the RNA quality meets the requirements

And (3) detecting the RNA degradation degree in the RNA sample after the DNA is removed by using 1% agarose gel electrophoresis, determining whether the sample is polluted, and displaying that the RNA of the sample is obviously degraded. The purity of the RNA was then determined using an ultraviolet spectrophotometer with an OD260/280 ratio of 2.37. RNA integrity was then analyzed by an Agilent 2100 bioanalyzer, which showed poor RNA integrity and a RIN of 4.5.

(S4) removal of rRNA

195 DNA probes with the same mass as 50bp complementary to rRNA sequences in the FFPE sample are designed and mixed together to form a DNA probe library, and then an experimental system shown as the following is prepared:

DNA Probe library 5. mu.g

DNA-depleted RNA sample 5. mu.g

5×hybridization buffer 5μL

(the composition was 1M NaCl, 0.5M Tris-HCl, pH7.4)

DEPC water was added to 25. mu.L.

The above test system was mixed at 95 ℃ for two minutes, and then the temperature of the test system was lowered to 45 ℃ at a rate of 0.1 ℃/s to form a DNA-RNA hybrid. Then 10. mu.L of RNase H (5U/. mu.L) and 5. mu.L of 10 XRNase H Reaction Buffer (composition: 500mM Tris-HCl, 750mM KCL, 30mM MgCl) preheated to 37 ℃ were added₂100mm dtt, pH 8.3) and 5 μ L DEPC water were added to the DNA-RNA hybrids. The RNA hybridized to the DNA was then digested at 37 ℃ for 30min and purified using 2.2 × RNA Clean XP beads. Then, 5. mu.L of LTurbo DNase I (2U/. mu.L) was added to the reaction system and the DNA probe was digested at 37 ℃ for 30 min. Subsequent use of 2.2 × RNA CleanXPbeads were purified to obtain rRNA depleted RNA, and the concentration of rRNA depleted was determined using a Qubit fluorometer, showing a concentration of 19.9 ng/. mu.L.

(S5) removal of Linear RNA

An experimental system was prepared as follows:

the above system was digested in a 37 ℃ water bath for 30min to obtain linear RNA-depleted RNA, and the concentration thereof was measured with a Qubit fluorometer, showing a concentration of 3.25 ng/. mu.L.

(S6) construction of circular RNA libraries for high throughput sequencing

(S61) fragmentation of RNA

An experimental system was prepared as follows:

5×First Strand Buffer 8μL

linear RNA-depleted RNA 10. mu.L

Nuclease free H₂O 2μL；

And (3) putting the experimental system into a PCR instrument for incubation, wherein the procedure of the PCR instrument is as follows: after 5min at 94 ℃, the RNA was immediately placed on ice to obtain fragmented RNA.

(S62) first strand synthesis:

an experimental system was prepared as follows:

and (3) putting the experimental system into a PCR instrument for incubation, wherein the procedure of the PCR instrument is as follows: 3min at 65 ℃ then placed on ice and 4. mu.L of nucleic free H was added₂O, 1. mu.L of 100mM DTT, 0.1. mu.L of 25mM dNTPs, 0.5. mu.L of SupeRase-In, 0.5. mu. L M-MulVReverse Transcriptase, and 4. mu.g of Actinomycin D. The reaction was then incubated in a PCR instrument programmed to: 10min at 25 ℃; 50min at-42 ℃; 15min at-70 ℃; hold at-4 ℃. Then 38. mu.L of RNA Clean XP and 19. mu.L of 100% ethanol were added for purification and 16. mu.L of nucleic Free H was used₂O, eluting to obtain RNA/cDNA hybrid, and transferring to new tube.

(S63) second Strand Synthesis

An experimental system was prepared at 0 ℃ as follows:

the above experimental system was incubated at 16 ℃ for 2.5 h. Subsequently 38. mu.L of RNA Clean XP and 19. mu.L of ethanol were added for purification and eluted with 32. mu.L of Qiagen EB to give dsDNA.

(S64) end repair

An experimental system was prepared at 0 ℃ as follows:

the above experimental system was placed in a PCR instrument and maintained at 20 ℃ for 30 min. Followed by addition of 28. mu.L of RNA CleanXP and 14. mu.L of ethanol and application of 17. mu.L of nucleic Free H₂And eluting to obtain the dsDNA after the end is repaired.

(S65) adding A tail

An experimental system was prepared as follows:

then incubated at 37 ℃ for 30 min. Followed by 28. mu.L of AMPure XP beads and 14. mu.L of ethanol and 10. mu.L of nucleic Free H₂O elution to obtain dsDNA with an A tail added.

(S66) Y-type Adapter connection:

an experimental system was prepared at 0 ℃ as follows:

the above assay system was placed in a PCR instrument and incubated at 20 ℃ for 20min, followed by mixing with 24. mu.L of "12P XP" beadsIncubate at room temperature for 6min in combination, after which the supernatant is retained and mixed with 12. mu.L of AMPure XP beads and 5. mu.L of 40 wt% PEG8000, incubate at room temperature for 6min, followed by 10. mu.L of nucleic Free H₂O elution twice to give an eluent, after which the eluent was mixed with 12 μ L AMPure XP, incubated again at room temperature for 6min, and then eluted 1 time using 30 μ L qiagen eb to give the product.

(S67) second Strand digestion and library amplification

mu.L of the above product was mixed with 1. mu.L of Uracil DNA Glycosylase and incubated at 37 ℃ for 30min to give UNG digested DNA. The experimental system shown below was then formulated at 0 ℃:

performing near-PCR amplification on the experimental system under the following cycle conditions: 30s at 94 ℃; (98 ℃ for 10 s; 65 ℃ for 30 s; 72 ℃ for 30s) for 15 cycles; 5min at 72 ℃; the temperature of the experimental system was then maintained at 4 ℃. Subsequently, 43. mu.L of AMPure XPbeads were added for purification and eluted using 12. mu.L of Qiagen EB to generate a library.

(S68) quality control and preparation of an indexed library

The concentration of the library was first determined using a Qubit fluorometer, resulting in 5.24 ng/. mu.L. Then, the Agilent 2100 bioanalyzer is used for detecting the quality of the library, the detection result is shown in figure 2, the size of the insert is qualified, the peak type is single, and the peak value is about 200-500 bp. Different indexing libraries are then mixed according to the detected concentration. And (4) detecting the concentration of the library by using a Qubit fluorometer, and then starting to continue the machine sequencing work.

Sequencing example bioinformatics analysis of sequencing data

The circular RNA high-throughput sequencing library constructed in examples 1-3 and the circular RNA high-throughput sequencing library constructed by a literature report method (see molecular cell 56, 55-66, October 2,2014) were sequenced on the Illumina sequencing platform. The results are shown in Table 1.

TABLE 1 bioinformatic analysis of sequencing results of the methods of the invention and literature reporting methods

The results of sequencing data analyzed by bioinformatics are shown in table 1, and by contrast, the method provided by the embodiment of the invention can further remove rRNA in a sample, the residual ratio of rRNA in the data is less than 0.1%, while the residual ratio of rRNA in a literature report method is 5%, for RNA samples with serious degradation such as FFPE and the like, the residual ratio is as high as 37%, the data amount is seriously wasted, and the method provided by the invention can obviously improve the effective sequencing data amount of circular RNA. In terms of the detection quantity of circular RNA, the circular RNA detected by a sample (cell sample) with good RNA integrity by using a literature report method is more, but the sample with poor RNA quality, particularly an FFPE sample, is remarkably superior to the literature report method. In addition, the method for constructing the circular RNA high-throughput sequencing library has the advantages of standard operation flow, optimized conditions, stable result and low cost which is only 1/3 of a method reported in a literature. 20 circular RNAs are randomly selected from the sequencing result to carry out QPCR verification, and the positive rate of the verification result is 100% (figure 3), thereby powerfully confirming the reliability of the method.

Therefore, the method provided by the embodiment of the invention can obviously further enrich the circular RNA in the sample, greatly reduce the data volume of circular RNA sequencing, reduce the cost and reduce the waste of manpower and material resources.

Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims (2)

1. A construction method of a circular RNA high-throughput sequencing library is characterized by sequentially comprising the following steps:

s1 extracting total RNA in the sample;

s2 removing DNA in the sample;

s3, detecting and evaluating the total RNA quality in the sample, and determining that the RNA quality meets the requirement;

s4 removing rRNA;

s5 removal of linear RNA;

s6 constructing a circular RNA library for high-throughput sequencing;

the step S4 of removing rRNA sequentially comprises the following steps:

s41, DNA probes complementary to the rRNA sequences are mixed in equal mass to form a DNA probe library;

s42, mixing and hybridizing the obtained DNA probe library with RNA which is detected and evaluated to determine the quality of the RNA according with the requirement, and forming a DNA-RNA hybrid;

s43 digesting the obtained DNA-RNA hybrid with RNase H;

s44 digesting the DNA probe with DNase I to obtain rRNA depleted RNA;

s45 determining the concentration of rRNA depleted RNA by fluorometry;

the step S3 of detecting the total RNA quality in the sample sequentially comprises the following steps:

s31 detecting the RNA degradation degree and whether there is pollution by 1% agarose gel electrophoresis;

s32, detecting the purity of RNA, namely the OD260/280 ratio, by using an ultraviolet spectrophotometer;

s33 testing the integrity of the RNA;

the weight ratio of the DNA probe library to the RNA used in the step S42 is (1-2): 1;

the conditions for hybrid hybridization in the step S42 are hybridization at a temperature of 95 ℃ for 2 minutes, and then the temperature is slowly decreased to 45 ℃ at a speed of 0.1 ℃/S;

the conditions for digesting the obtained DNA-RNA hybrid with RNase H in the step S43 are digestion at a temperature of 37 ℃ for 30 minutes;

the construction of the circular RNA library for high-throughput sequencing in the step S6 sequentially comprises the following steps:

s61RNA fragmentation;

s62 first strand synthesis: the method is carried out by adopting a method of reverse transcriptase and random primers;

second strand synthesis of S63: digesting an RNA strand in the RNA/cDNA hybrid by RNase H, and then synthesizing a second strand by DNA polymerase I;

s64 end repair: using End Repair Mix to form blunt-ended dsDNA and adding phosphate group at 5' End;

s65 with a tail: add A tail to dsDNA ends using Klenow 3'-5' exo-;

S66Y Adapter connection: t4 DNA Ligase is used for connecting the Adapter and the dsDNA with an A tail added;

s67 second strand digestion and library amplification: digesting the second strand with UNG enzyme;

s68 quality inspection and preparation of an indexing library;

the condition for RNA fragmentation in step S61 is fragmentation at a temperature of 90-96 ℃ for 3-6 minutes.

2. A kit for constructing a circular RNA high-throughput sequencing library, which is characterized by comprising:

(1) cyclic RNA enrichment reagent, DNase I, RNase H and RNaseR;

(2) species-specific DNA probes;

(3) various enzymes required for library construction: DNA polymerase I, Large Fragment, T4 PolynucleotideKinase, Escherichia coli UDG, T4 DNA Ligase, and Phusion High-Fidelity DNApolymerase;

(4) magnetic beads, dNTP, nucleic Free Water, a joint and a primer,

wherein the species-specific DNA probe is complementary to an rRNA sequence of the corresponding species.

Images