WO2023065615A1 - Probe for blocking ribosome rna or globulin rna in rna library construction process, and use thereof - Google Patents

Abstract

Provided are a probe for blocking ribosome RNA or globulin RNA in an RNA library construction process, and the use thereof in the rapid removal of ribosome RNA or globulin RNA in RNA library construction. The length of the probe is 35-60 nt; the probe and ribosome RNA or globulin RNA form a ternary blocking complex by means of complementary pairing; the second half region of the probe and target RNA strictly form complementary pairing, and 5-9 bases at the 5' end of the pairing region of the probe are replaced with modified bases; and the first half region of the probe comprises pyrimidine bases and is used for being inserted into the major groove of the two strands of DNA/RNA to form a ternary complex. The 3'-OH of the probe is blocked by means of MGB so as to prevent extension with the probe as a primer; moreover, the MGB can be inserted into the minor groove of the two strands of DNA/RNA, so that the stability of the ternary complex is improved.

Description

Probe for blocking ribosomal RNA or globulin RNA in the process of RNA library construction and its application technical field

The patent of this application relates to a probe for blocking ribosomal RNA or globulin RNA in the process of building an RNA library, which belongs to the field of biotechnology.

Background technique

In recent years, with the rapid development of high-throughput sequencing technology, various RNA library construction and sequencing technologies have emerged and iteratively upgraded, greatly reducing the difficulty and cost of RNA library construction and sequencing, which has also made RNA high-throughput sequencing Technology has become an important research tool in fields such as life progression and disease diagnosis. However, the proportion of RNA species in samples from different sources is extremely heterogeneous. For example, ribosomal RNA (rRNA) accounts for about 90%-95% of the total RNA in normal cells, and globulin mRNA accounts for about 95% of the total RNA in blood. More than 76% of mRNA, the presence of these RNAs occupies most of the sequencing data, which seriously affects the detection of low-abundance RNAs and increases the cost of sequencing.

There are currently two main types of strategies to remove these high-abundance RNAs that are not needed during RNA library construction. The first type of strategy is magnetic bead capture. For example, the polyA enrichment method uses oligo(dT) magnetic beads to capture and enrich mRNA containing polyA. This method is only suitable for RNA with better integrity, and the types of RNA that can be captured are limited, which greatly reduces the diversity of sequencing. Moreover, this method has a higher capture efficiency for RNA with longer polyA length, which will cause a preference for RNA library construction; or target RNA capture and removal method, after hybridizing with a biotin-modified antisense DNA probe and target RNA, Target RNA was removed using streptavidin magnetic beads. Another type of strategy is nuclease degradation. For example, the RNase H cleavage removal method uses RNase H to specifically recognize and degrade the activity of RNA in the DNA:RNA hybrid strand to degrade the target RNA; or the CRISPR/Cas9 cleavage method uses the sgRNA-Cas9 system to degrade the constructed double-stranded DNA library. The double-stranded DNA derived from the targeted RNA is cleaved. These methods have defects such as high cost, complicated operation (about 20-40 steps), long time-consuming (about 2 hours), high requirements on sample source and RNA abundance, and difficult storage of reagents, which seriously hinder the development of RNA next-generation sequencing library construction technology. Development of industrial automation and its application in the field of rapid diagnosis of diseases.

We have previously developed a rapid hybridization technique that blocks reverse transcription of target RNA by using a probe to form a double-stranded complex with the target RNA, thereby blocking the reverse transcription of the target RNA. Using this technology, we successfully and efficiently reduced the proportion of ribosomal RNA and globulin RNA sequencing data in the process of RNA library construction (202110257924.X; 202110553161.3). However, this method has strict requirements on the performance of reverse transcriptase. For reverse transcriptase with high strand displacement ability, the removal effect of this method is not good, because the reverse transcriptase with high strand displacement ability will untangle the hybridization probe. The double helix formed with the target RNA leads to a decrease in the reverse transcription inhibition effect of the target RNA. For RNAs with high GC regions (such as exon regions), the strand displacement activity of reverse transcriptase is the key to ensure efficient reverse transcription of these regions. Therefore, there are still application limitations in using this method to remove ribosomal RNA and globulin RNA during RNA library construction.

Contents of the invention

This application provides a probe for blocking ribosomal RNA or globulin RNA in the process of RNA library construction. The probe is 35-60 nt in length and consists of a chimeric region in the first half and a pairing region in the second half. The chimeric region is a pyrimidine base, the pairing region is strictly complementary to the target RNA, and the first 5-9 bases of the pairing region are replaced by modified bases, and the 3'-OH of the probe is replaced by a small groove Binder (MGB) for blocking.

Alternatively, the application provides a probe for blocking ribosomal RNA or globulin RNA during RNA library construction, wherein,

The probe length is 35-60nt;

According to the 5' to 3' direction, the probe comprises a chimeric region and a pairing region, wherein the chimeric region consists of pyrimidine bases, the pairing region is strictly complementary to the target RNA, and 5 of the pairing region 5-9 bases at the ' end are modified; and

The 3'-OH of the probe was blocked with MGB.

In some embodiments, 5-9 bases at the 5' end of the paired region are covered with peptide nucleic acid (PNA), locked nucleic acid (LNA), deoxyuridine (dU), 2-O-methylribose, 5-hydroxy Butyrate acyl-deoxyuridine, 2-aminodeoxyadenosine or 5-methyldeoxycytidine modification.

In some embodiments, 5 bases at the 5' end of the pairing region are modified. In other embodiments, the 6 bases at the 5' end of the pairing region are modified. In other embodiments, the 7 bases at the 5' end of the pairing region are modified. In other embodiments, the 8 bases at the 5' end of the pairing region are modified. In other embodiments, the 9 bases at the 5' end of the pairing region are modified. In such embodiments, it is calculated from 1 base at the 5' end of the paired region. For example, the modification of 5 bases at the 5' end of the pairing region means that the 1 to 5 bases at the 5' end of the pairing region are modified.

In some embodiments, the chimeric and pairing regions are the same length.

In some embodiments, a TAT sequence is also included between the chimeric region and the pairing region to form a hairpin structure.

In some embodiments, the probe provided herein has the sequence shown in any one of SEQ ID NO: 14 to SEQ ID NO: 159.

Preferably, there are multiple probes, the probes cover ribosomal RNA or globulin RNA region not less than 1/6 of the full-length RNA of ribosomal RNA or globulin RNA, and the probes cover ribosomal RNA or globulin RNA Evenly distributed on the surface, the distance between the probes is not more than 100nt, the modification types of the modified bases are PNA, LNA, dU, 2-O-methyl ribose, 5-hydroxybutyryl-deoxyuridine, 2 -Aminodeoxyadenosine or 5-methyldeoxycytidine modification.

Alternatively, the application provides a mixture of probes that block ribosomal RNA or globulin RNA during RNA library construction, wherein,

The mixture contains a plurality of probes, the plurality of probes covering ribosomal RNA or globulin RNA region is no less than 1/6 of the ribosomal RNA or globulin RNA full-length RNA, and the distance between the probes is no more than 100nt ;

and where:

Each probe is 35-60nt in length;

According to the 5' to 3' direction, each probe comprises a chimeric region and a pairing region, wherein the chimeric region consists of pyrimidine bases, the pairing region is strictly complementary to the target RNA, and 5 of the pairing region 5-9 bases at the 'end are modified by PNA, LNA, dU, 2-O-methylribose, 5-hydroxybutyryl-deoxyuridine, 2-aminodeoxyadenosine or 5-methyldeoxycytidine ;and

The 3'-OH of each probe was blocked with MGB.

Preferably, the ribosomal RNA is people's 5.8S rRNA, and the probe is a mixture of the 5.8S probe shown in the table below:

Taking 5.8S probe-1 as an example, TCCCCTCCTCCCTCTT (SEQ ID NO: 175) is a chimeric region, TAT forms a hairpin structure, and ATCGACGCACGAGCCGA (SEQ ID NO: 176) is a pairing region.

Preferably, the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.

Preferably, the ribosomal RNA is people's 18S rRNA, and the probe is a mixture of the 18S probe shown in the table below:

Preferably, the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.

Preferably, the ribosomal RNA is people's 28S rRNA, and the probe is a mixture of the 28S probe shown in the table below:

Preferably, the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.

Preferably, the ribosomal RNA is people's 12S rRNA, and the probe is a mixture of 12S probes shown in the table below:

Preferably, the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.

Preferably, the ribosomal RNA is people's 16S rRNA, and the probe is a mixture of 16S probes shown in the table below:

Preferably, the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.

Preferably, the probe is a mixture of Globin probes shown in the table below:

Preferably, the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.

The application of the above-mentioned probes to quickly remove ribosomal RNA or globulin RNA in RNA library construction.

Preferably, the steps include:

(1) Extract the total RNA in the sample, add probes to the total RNA, and anneal after high-temperature denaturation;

(2) adding a reverse transcription buffer and a cDNA synthetase mixture to the reaction product to carry out a reverse transcription reaction;

(3) RNA NGS library construction.

The binary reverse transcription probe disclosed in CN202110257924.X forms a double helix with the target RNA, and the double helix structure will be unraveled when encountering a reverse transcriptase with helicase activity so that reverse transcription cannot be hindered. The application provides a probe and method for sealing ribosomal RNA and globulin RNA in the process of RNA library construction. ). The length of the probe is 35-60nt, the second half region of the probe is strictly complementary to the target RNA, and the 5-9 bases at the 5' end of the probe pairing region are replaced by modified bases to enhance the binding strength to the target RNA and ability; the first half region of the probe is a pyrimidine base, which is used to fit into the major groove of the DNA/RNA double strand to form a ternary complex. The 3'-OH of the probe is blocked by MGB to prevent the probe from being extended as a primer; at the same time, MGB can be chimerized to the minor groove of the DNA/RNA double strand to improve the stability of the ternary complex. The base of the polypyrimidine region of the ternary complex probe of the present application is small, and can be fitted into the major groove of the DNA double helix region, and the MGB modification can be fitted into the minor groove of the double helix, thereby forming a stable triplex. As a result of the metacomplex, the helicase cannot efficiently unwind this structure compared to the binary RT probe. Therefore, the binding of the ternary complex probe to the target RNA is more efficient and stable, and the hindrance effect on reverse transcription is stronger. This application uses the ternary complex blocking probe method to achieve high-efficiency and high-specificity inhibition of ribosomal RNA and globulin RNA reverse transcription during the one-strand cDNA synthesis process of RNA library construction, thereby hindering ribosomal RNA and globulin RNA Participate in the downstream library building process. The application has the advantages of simple operation, extremely short time consumption, strong specificity, wide applicability, high removal efficiency and high number of gene detection, and is very suitable for industrial automation database construction and rapid disease diagnosis.

Description of drawings

Figure 1 is a schematic diagram of the reverse transcription hindering probe method.

Figure 2 is a schematic diagram of the inhibition of reverse transcription synthesis of 5.8S rRNA by the reverse transcription hindering probe.

Figure 3 is the qPCR method to identify the inhibitory effect of different modified types of probes on reverse transcription.

Figure 4 shows the inhibitory effect of the distribution position of PNA in the probe identified by qPCR on the reverse transcription of target RNA.

Figure 5 is a qPCR method to identify the inhibitory effect of PNA modified probes with different lengths on target RNA reverse transcription.

Fig. 6 is a schematic diagram of the principle of full-coverage reverse transcription hindering probe competition for binding to target RNA.

Figure 7 is a qPCR method to identify the inhibitory effect of the full coverage reverse transcription hindering probe on the synthesis of 5.8S rRNA reverse transcription.

Figure 8 is a qPCR method to identify the inhibitory effect of the full-coverage reverse transcription hindering probe on rRNA reverse transcription synthesis.

Figure 9 is a schematic flow chart of five rRNA removal methods in the process of NGS RNA library construction.

Figure 10 shows the effect of qPCR verification of five rRNA removal methods.

Figure 11 shows the RNA library yields of five methods for removing rRNA.

Figure 12 compares the effect of five rRNA removal methods by RNA-seq.

Figure 13 shows the sequencing correlation of the six library construction methods.

Figure 14 shows the number of genes detected by the six library construction methods at the same sequencing depth.

Figure 15 is a schematic diagram of the application process of the ternary complex blocking probe on Nanopore Direct cDNA sequencing.

Figure 16 shows the effect of the ternary complex blocking probe on the proportion of RNA in Nanopore Direct cDNA sequencing data.

Figure 17 shows the effect of the ternary complex blocking probe on the number of genes detected in the Nanopore Direct cDNA sequencing data.

Figure 18 is the removal effect of the ternary complex blocking probe on rRNA and Globin mRNA in blood RNA detection.

Figure 19 shows the effect of the ternary complex blocking probe on the number of gene detection in blood RNA detection.

Figure 20 is a performance comparison of the ternary complex blocking probe and the double-stranded reverse transcription blocking probe.

Detailed ways

The probe and primer sequences and modifications used in the following examples are shown in Table 1. N is a random base, that is, any base in A, T, C, and G, and Random Hexamers is a general-purpose base used for reverse transcription. primers.

Table 1 Probe and primer sequences

The sequences 166-175 are quantitative primers for RT-qPCR.

Example

Example 1: Inhibitory effect of different modification types of probes on target RNA reverse transcription

Referring to Fig. 2, the present embodiment designs 5.8S-specific probes (Table 1 No. 1-5) with different modifications to suppress the 5.8S rRNA reverse transcription process. The specific implementation is as follows:

Table 2

components	Dosage
Total RNA		1μg
1 μM 5.8S modified probe (any one of No. 1-5 in Table 1)	1μL
1μM 5.8S rRNA RT primer	1μL
10mM dNTPs mix (with 0.5M KCl)	1μL
Supplement DEPC H2O to	13μL

Mix and spin away. React at 75°C for 1 min and store at 4°C.

table 3

Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C;

After diluting 1000 times, Hieff

qPCR SYBR Green Master Mix (11200ES03) was used to quantify 5.8S cDNA, and the quantitative primers are listed in Table 1. Quantitative results are shown in Figure 3.

From the quantitative results, it can be seen that peptide-containing nucleic acid (PNA), locked nucleic acid (LNA), deoxyuracil (dU), DNA minor groove binder (MGB) and 2-oxomethyl (2'-O-Me) modification All the ternary complex probes can effectively inhibit the reverse transcription of target RNA, and the PNA probe has the best inhibitory effect on reverse transcription (see Table 4).

Table 4: Inhibition efficiency of target RNA reverse transcription with different 5.8s rRNA probe inputs

Probe modification type	undecorated	PNA	LNA	U	MGB	2'-O-Me
Reverse transcription inhibition efficiency (%)	75.35	99.97	99.93	91.18	89.36	84.15

Example 2: The inhibitory effect of the distribution position of PNA in the probe on the reverse transcription of target RNA

In this embodiment, 5.8S-specific probes (mixture of sequences No. 6-8 in Table 1) of different PNA modification positions are designed to inhibit the reverse transcription process of 5.8S rRNA. DETAILED DESCRIPTION Referring to Example 1, the quantitative results are shown in FIG. 4 .

It can be seen from the quantitative results that when the position of the PNA in the probe is close to the left end (ie, the 5' end) of the pairing region between the probe and the target RNA, the effect of hindering the reverse transcription of the target RNA is higher than that when the position of the PNA in the probe is close to the probe. The right end of the needle (i.e. the 3' end).

Table 5: Inhibition efficiency of target RNA reverse transcription by PNA distribution position in the probe

Distribution of PNA modification positions in probes	Left	Middle	Right
Reverse transcription inhibition efficiency (%)	98.94	95.13	86.81

Example 3: Inhibitory effect of different length PNA modified probes on target RNA reverse transcription

In this example, 5.8S-specific probes with PNA modifications of different lengths (the mixture of sequence numbers 9-14 in Table 1) were designed to inhibit the reverse transcription process of 5.8S rRNA. DETAILED DESCRIPTION Referring to Example 1, the quantitative results are shown in FIG. 5 .

It can be seen from the quantitative results that when the pairing length of the PNA probe and the target RNA reaches 15-20 nt, the target RNA can be efficiently inhibited by reverse transcription.

Table 6: Inhibition efficiency of target RNA reverse transcription by PNA modified probes with different lengths

Different length PNA probes	15nt	20nt	25nt	30nt	35nt	40nt
Reverse transcription inhibition efficiency (%)	98.39	98.57	98.62	98.68	98.77	98.72

Example 4: 5.8S full-coverage reverse transcription hinders the competition of random primer binding and the inhibition of one-strand cDNA synthesis

Referring to Fig. 6, this embodiment utilizes the characteristics of high Tm value and high binding capacity of the 5.8S-specific PNA modified probe (the mixture of sequence numbers 15-17 in Table 1) to achieve efficient and specific inhibition of 5.8S rRNA reverse transcription. The specific implementation is as follows:

Table 7

components	Dosage
Total RNA	1μg
0.5-8μM 5.8S probe mix	1μL
50μM Random Hexamers	1μL
10mM dNTPs mix (with 0.5M KCl)	1μL
Supplement DEPC H2O to	13μL

Mix and spin away. React at 75°C for 1 min, store at 4°C;

Table 8

Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C;

After diluting 1000 times, Hieff

qPCR SYBR Green Master Mix (11200) was used to quantify 5.8S, 18S, 28S, Actin and Gadph cDNA. The quantitative primers are shown in Table 1, and the quantitative results are shown in Figure 7.

It can be seen from the quantitative results that the 5.8S probe mixture designed by this method can efficiently and specifically inhibit the reverse transcription of target RNA, and has little effect on the reverse transcription of other non-target RNAs (GAPDH or ACTB mRNA). When the amount of probe input reaches 1 μM, the reverse transcription inhibition efficiency of 5.8S rRNA can reach more than 99%.

Example 5: 18S, 28S, 12S and 16S rRNA full coverage reverse transcription hinders the competition of random primer binding and the inhibition of one-strand cDNA synthesis

This embodiment has verified 18S rRNA (the mixture of sequence number 17-47 in table 1), 28S rRNA (the mixture of sequence number 48-111 in table 1), 12S rRNA (the mixture of sequence number 112-122 in table 1) and 16S rRNA (The mixture of sequence numbers 123-139 in Table 1) inhibits efficiency and specificity of target RNA reverse transcription. The probe sequences and primers are shown in Table 1, and the quantitative results are shown in Figure 8.

It can be seen from the quantitative results that the reverse transcription blocking probe designed in this method can efficiently block the reverse transcription of 18S, 28S, 12S and 16S rRNA, and the removal efficiency is as high as 99%.

Example 6: Comparison of methods for removing ribosomal RNA

Referring to Figure 9, this example systematically compares the removal effects of polyA RNA enrichment, RNase H cleavage and removal, hybridization capture and removal, CRISPR cleavage and removal, and ternary complex blocking probe methods on target RNA. The specific implementation is as follows:

1) polyA enrichment method. Use Hieff

MaxUp II Dual-mode mRNA Library Prep Kit for Illumina (Yeasen, 12300) was used to enrich mRNA.

Take 1 μg of total RNA and make up the volume to 50 μL with Nuclease free water. Pipette 50 μL mRNA Capture Beads into 50 μL total RNA sample, mix by pipetting. 5min at 65°C; incubate at room temperature for 5min. Place the sample in a magnetic stand, let it stand at room temperature for 5 min, and carefully remove the supernatant. Take the sample out of the magnetic stand, resuspend the magnetic beads with 200 μL of Beads Wash Buffer, and mix by pipetting repeatedly. Place the sample in a magnetic stand, let it stand at room temperature for 5 min, and carefully remove the supernatant. Repeat washing the beads once. Add 50 μL Tris Buffer to resuspend the magnetic beads, and mix well by pipetting. 80°C for 2min; store at 25°C. Take the sample out of the PCR machine, add 50 μL of Beads Binding Buffer, and mix by pipetting repeatedly. Place at room temperature for 5 minutes. Place the sample in a magnetic stand, let it stand at room temperature for 5 min, and carefully remove the supernatant. Resuspend the magnetic beads with 200 μL of Beads Wash Buffer, pipette and mix well, put the sample back into the magnetic stand, let it stand at room temperature for 5 minutes, and suck off all the supernatant. Take the sample out of the magnetic stand, resuspend the magnetic beads with 18.5 μL Frag/Prime buffer, and mix well by pipetting. Place the sample in a PCR instrument at 94°C for 5 minutes, and immediately place it on a magnetic stand. Aspirate 17 μL of fragmented mRNA, add 6 μL Strand Specificity Reagent and 2 μL 1st Strand Enzyme Mix, mix well and spin off. 10min at 25°C, 15min at 42°C, 15min at 70°C, and store at 4°C. After 0.5 μL of the product was diluted 100 times, Hieff

qPCR SYBR Green Master Mix (11200) quantifies 5.8S, 18S, 28S and Actin cDNA.

2) RNase H cut removal method. Use Hieff

MaxUp rRNA Depletion Kit (Human/Mouse/Rat) (Yeasen, 12253) removes rRNA.

Table 9

components	Dosage
Total RNA	1μg
Hybridization Buffer	3μL
Probe Mix(H/M/R)	1μL
Supplement DEPC H2O to	15μL

Mix by pipetting and centrifuge immediately. React at 95°C for 2 minutes, react at 95°C-22°C-0.1°C/s, react at 22°C for 5 minutes, and store at 4°C.

Table 10

components	Dosage
The above reaction system	15μL
RNase H buffer	3μL
RNase H	2μL
total capacity	20 μL

After mixing by blowing and blowing, centrifuge immediately, react at 37°C for 30min, and store at 4°C.

Table 11

After mixing by blowing and blowing, centrifuge immediately, store at 37°C for 30min, and store at 4°C. Add 110 μL Hieff

RNA Cleaner (Yeasen, 12602), thoroughly mixed by pipetting, and incubated at room temperature for 5 minutes. Place the PCR tube in a magnetic stand to separate the magnetic beads and the liquid. After the solution is clarified (about 3 min), carefully remove the supernatant. Keep the PCR tube in the magnetic stand all the time, add 200 μL Nuclease free H2O freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant. Repeat rinse once. Aspirate the remaining liquid with a 10 μL pipette. Keep the PCR tube in the magnetic stand all the time, open the cover and dry the magnetic beads at room temperature (5-10min). Resuspend the magnetic beads with 18.5 μL Frag/Prime buffer, mix by pipetting, and let stand at room temperature for 5 minutes. Place the PCR tube in a magnetic stand to separate the magnetic beads and the liquid. After the solution is clarified (about 3 min), pipette 17 μL of mRNA into a new PCR tube. Place the sample in a PCR instrument at 94°C for 5 minutes, then immediately ice-bath for 5 minutes.

Table 12

components	Dosage
Frag/Prime buffer and RNA	17μL
Strand Specificity Reagent	6μL
1st Strand Enzyme Mix	2μL
total capacity	25 μL

Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C. After 0.5 μL of the product was diluted 100 times, Hieff

qPCR SYBR Green Master Mix (11200) quantifies 5.8S, 18S, 28S and Actin cDNA.

3) Hybrid capture depletion method: use Illumina's Ribo-Zero Plus rRNA Depletion Kit for rRNA depletion, and the operation procedure follows the instructions.

4) Ternary complex blocking probe method.

Table 13

Mix by pipetting and centrifuge immediately. React at 95°C for 5 minutes, react at 75°C for 1 minute, and store at 4°C.

Table 14

components	Dosage
The above reaction system	17μL
Strand Specificity Reagent	6μL
1st Strand Enzyme Mix	2μL
total capacity	25 μL

Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C. After 0.5 μL of the product was diluted 100 times, Hieff

qPCR SYBR Green Master Mix quantifies 5.8S, 18S, 28S and Actin cDNA.

5) CRISPR cutting removal method: using TAKARA's

Stranded Total RNA-Seq Kit was used to remove rRNA, and the operation procedure was in accordance with the instructions.

Quantitative results are shown in Figure 10. From the quantitative results, it can be seen that the ternary complex blocking probe can effectively inhibit the first-strand synthesis efficiency of rRNA during the process of NGS RNA library construction, and the removal effect reaches more than 99%, which has significantly exceeded the RNase H cutting removal method and hybridization capture removal method. The removal efficiency of the method and the CRISPR cleavage removal method is comparable to that of the polyA enrichment method, and has higher specificity, and does not affect the first-strand synthesis of other non-target RNAs.

In addition, the remaining 1st strand cDNA product was passed through Hieff

MaxUp II Dual-mode mRNA Library Prep Kit for Illumina (Yeasen, 12300) was used to build the library, and the process included:

1) Second strand synthesis

Table 15

components	Dosage
1st strand cDNA	24 μL
DEPC H2O	1μL
2nd Strand Buffer	30μL
2nd Strand Enzyme Master Mix	5μL
total capacity	60μL

Mix by pipetting and centrifuge immediately. React at 16°C for 30 minutes, react at 72°C for 15 minutes, and store at 4°C.

2) Joint connection

Table 16

components	Dosage
dA-tailed DNA	60μL
Ligation Enhancer	30μL
Quick T4 DNA Ligase	5μL
DNA Adapter	5μL
total capacity	100μL

Mix by pipetting and centrifuge immediately. React at 20°C for 15 minutes and store at 4°C.

3) Two rounds of magnetic bead recovery

Add 60 μL Hieff

DNA Selection Beads (Yeasen, 12601), thoroughly mixed by pipetting, and incubated at room temperature for 5 minutes. Place the PCR tube in a magnetic stand to separate the magnetic beads and the liquid. After the solution is clarified (about 3 min), carefully remove the supernatant. Keep the PCR tube in the magnetic stand all the time, add 200 μL Nuclease free H2O freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat rinse once. Aspirate the remaining liquid with a 10 μL pipette. Keep the PCR tube in the magnetic stand at all times, open the cover and dry the magnetic beads at room temperature (5min). Add 53 μL ddH2O, pipette until fully mixed, and let stand at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it in a magnetic stand. After the solution is clarified (about 5 min), carefully pipette 50 μL of the supernatant into a new PCR tube.

Add 80 μL Hieff

For DNA Selection Beads, repeat the recovery operation above. Add 22 μL ddH2O, pipette until fully mixed, and let stand at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it on a magnetic stand. After the solution is clarified (about 5 min), carefully pipette 20 μL of the supernatant into a new PCR tube.

4) Library amplification, recovery, quantification and sequencing

Table 17

Mix by pipetting and centrifuge immediately. Library amplification was performed according to the following reaction procedure.

Table 18

Add 45 μL Hieff

For DNA Selection Beads, repeat the recovery operation above. Add 22 μL ddH2O, pipette until fully mixed, and let stand at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it on a magnetic stand. After the solution is clarified (about 5 min), carefully pipette 20 μL of the supernatant into a new PCR tube. The library was quantified with the dsDNA HS Assay Kit for Qubit (Yeasen, 12640), and the library was sequenced using the Illumina NovaSeq 6000 platform.

It can be seen from Figure 11 and Figure 12 that, compared with the other four existing methods, the ternary complex blocking probe method can significantly increase the yield of RNA library construction, and the removal effect on ribosomal RNA is more effective, which shows that the reversal The recording-blocking probe method can efficiently and quickly remove the target RNA during the library construction process without affecting the reverse transcription of other non-target RNAs with less loss. In addition, the results of correlation analysis (see Figure 13) showed that the ternary complex blocking probe method had a better correlation with RNA direct library construction, which indicated that the reverse transcription hindering probe method had less preference for library construction and was more efficient. Can reflect the true information of the original RNA. At the same time, at the same sequencing depth, the number of genes detected by the reverse transcription probe method is significantly higher than that of the other three library construction methods (see Figure 14), which indicates that the information obtained by the RNA sequencing technology combined with the reverse transcription probe method is more complete ,comprehensive.

Example 7: Application of ternary complex blocking probe in three-generation Nanopore sequencing.

In this example, the application of the ternary complex blocking probe on the third-generation Nanopore direct cDNA-seq was tested, as shown in Figure 15. The specific implementation is as follows:

Table 19

components	Dosage
Total RNA	2μg
10x E.coli poly(A)polymerase buffer	1μL
poly(A)polymerase buffer	1μL
10mM ATP	1μL
Supplement DEPC H2O to	10 μL

React at 37°C for 30 minutes. Add OligodT23VN and rRNA probe mix (the mixture of sequences No. 15, 18, 48, 112, and 123 in Table 1), store at 75°C for 1min, and store at 4°C. The library was constructed and sequenced according to Nanopore's Direct cDNA Sequencing Kit process.

The results are shown in Figure 16 and Figure 17, the use of RNA polyadenylation and ternary complex blocking probes can significantly reduce the proportion of rRNA source data in the sequencing data, and increase the number of genes detected by Direct cDNA Sequencing. This indicates that the ternary complex blocking probe has important application value in the third-generation Nanopore sequencing.

Example 8: Application of Globin RNA ternary complex blocking probe in blood RNA detection.

The present embodiment has designed Globin RNA ternary complex blocking probe (the mixture of sequence number 140-159 of table 1), and has detected its application on blood RNA detection, and specific implementation is as follows

Table 20

Mix by pipetting and centrifuge immediately. React at 95°C for 5 minutes, react at 75°C for 1 minute, and store at 4°C.

Table 21

components	Dosage
The above reaction system	17μL
Strand Specificity Reagent	6μL
1st Strand Enzyme Mix	2μL
total capacity	25 μL

Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C. RNA library construction and sequencing were performed according to the above-mentioned examples.

The results are shown in Figure 18 and Figure 19, the ternary complex blocking probe can significantly reduce the proportion of rRNA and Globin RNA in RNA sequencing data, and greatly increase the number of genes detected in blood RNA detection.

Example 9: Performance comparison of ternary complex blocking probe and double-stranded reverse transcription blocking probe.

This example compares the application effect of the ternary complex blocking probe of the present application and the double-stranded reverse transcription hindering probe (CN202110257924.X) on RNA, and the specific implementation method is as follows

Table 22

Mix by pipetting and centrifuge immediately. React at 95°C for 5 minutes, react at 75°C for 1 minute, and store at 4°C.

Table 23

components	Dosage
The above reaction system	17μL
Strand Specificity Reagent	6μL
40U/μL RNase inhibitor	1μL
200U/μL reverse transcriptase	1μL
total capacity	25 μL

Reverse transcriptases include Hifair II/III/IV/V Reverse transcriptase of Yisheng Biology.

Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C. RNA library construction and sequencing were performed according to the above-mentioned examples.

The results are shown in Figure 20. Compared with the double-stranded reverse transcription blocking probe, the ternary complex blocking probe has wider applicability to reverse transcriptase and higher rRNA removal efficiency in RNA library construction.

The application provides a method for sealing ribosomal RNA and globulin RNA in the process of RNA library construction. The principle is to use probes to form a triple complex (Triple complex) with ribosomal RNA and globulin RNA through complementary pairing. During the one-strand cDNA synthesis process of RNA library construction, high-efficiency and high-specificity inhibition of ribosomal RNA and globin RNA reverse transcription is achieved, thereby preventing ribosomal RNA and globin RNA from participating in the downstream library construction process. The application has the advantages of simple operation, extremely short time consumption, strong specificity, wide applicability, high removal efficiency and high number of gene detection, and is very suitable for industrial automation database construction and rapid disease diagnosis.

Claims (18)

1. A probe for blocking ribosomal RNA or globulin RNA during RNA library construction, wherein,

   The probe length is 35-60nt;

   According to the 5' to 3' direction, the probe comprises a chimeric region and a pairing region, wherein the chimeric region consists of pyrimidine bases, the pairing region is strictly complementary to the target RNA, and 5 of the pairing region 5-9 bases at the ' end are modified; and

   The 3'-OH of the probe was blocked with MGB.
2. The probe according to claim 1, wherein the 5-9 bases at the 5' end of the pairing region are replaced by PNA, LNA, dU, 2-O-methylribose, 5-hydroxybutyric acid acyl-deoxyuria Glycoside, 2-aminodeoxyadenosine or 5-methyldeoxycytidine modification.
3. The probe according to claim 1, wherein the probe has the sequence shown in any one of SEQ ID NO:14 to SEQ ID NO:159.
4. A mixture of probes that block ribosomal RNA or globulin RNA during RNA library construction, wherein the mixture contains multiple probes that cover ribosomal RNA or globulin RNA regions not less than 1/6 of the full-length RNA of ribosomal RNA or globulin RNA, the distance between the probes does not exceed 100nt;

   and where:

   Each probe is 35-60nt in length;

   According to the 5' to 3' direction, each probe comprises a chimeric region and a pairing region, wherein the chimeric region is composed of pyrimidine bases, the pairing region is strictly complementary to the target RNA, and 5 of the pairing region 5-9 bases at the 'end are modified by PNA, LNA, dU, 2-O-methylribose, 5-hydroxybutyryl-deoxyuridine, 2-aminodeoxyadenosine or 5-methyldeoxycytidine ;and

   The 3'-OH of each probe was blocked with MGB.
5. The mixture of probes according to claim 4, wherein: ribosomal RNA is people's 5.8S rRNA, and probe is the mixture of 5.8S probe shown in the table below:

   
6. The mixture of probes according to claim 5, wherein: the 3' end of the probe is blocked by MGB, and the italic bold base in the probe sequence is a PNA modified base.
7. The mixture of probes according to claim 4, wherein: the ribosomal RNA is people's 18S rRNA, and the probe is the mixture of 18S probe shown in the table below:

   

   

   
8. The mixture of probes according to claim 7, wherein: the 3' end of the probe is blocked by MGB, and the italic bold base in the probe sequence is a PNA modified base.
9. The mixture of probes according to claim 4, wherein: the ribosomal RNA is people's 28S rRNA, and the probe is the mixture of the 28S probe shown in the table below:

   

   

   

   
10. The mixture of probes according to claim 9, wherein: the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.
11. The mixture of probes according to claim 4, wherein: the ribosomal RNA is people's 12S rRNA, and the probe is the mixture of 12S probe shown in the table below:

    
12. The mixture of probes according to claim 11, wherein: the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.
13. The mixture of probes according to claim 4, wherein: the ribosomal RNA is people's 16S rRNA, and the probe is the mixture of 16S probe shown in the table below:

    

    
14. The mixture of probes according to claim 13, wherein: the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.
15. The mixture of probe according to claim 4, wherein: probe is the mixture of Globin probe shown in the table below:

    

    
16. The mixture of probes according to claim 15, wherein: the 3' end of the probe is blocked by MGB, and the bold bases in italics in the probe sequence are PNA modified bases.
17. Application of the probe according to any one of claims 1-3 or the mixture of probes according to any one of claims 4-16 in rapidly removing ribosomal RNA or globulin RNA in RNA library construction.
18. The use according to claim 17, the steps comprising:

    (1) extracting the total RNA in the sample, adding probes or a mixture of probes to the total RNA, annealing after high temperature denaturation;

    (2) Add reverse transcription buffer, random primer and one-strand cDNA synthetase mixture to the reaction product to carry out reverse transcription reaction;

    (3) RNA NGS library construction.

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