Construction method of extracellular trace free RNA high-throughput sequencing library and kit

Abstract

The invention discloses a construction method of an extracellular trace free RNA (Ribonucleic Acid) high-throughput sequencing library and a kit. The method comprises the following steps: extracting total RNA (Ribosomal Ribonucleic Acid) from extracellular fluid, fragmenting the RNA, closing ribosomal RNA (ribosomal Ribonucleic Acid) by using rRNA antigens, and reversely transcribing the RNA into cDNA (Complementary Deoxyribose Nucleic Acid) by using a random primer containing part of linker sequences, template replacement reverse transcriptase and template replacement oligonucleotide, so that the 3'end and the 5 'end of the synthesized cDNA contain part of linker sequences; then carrying out a first round of PCR pre-amplification, and adding a complete sequencing joint during a second round of PCR amplification; by means of the steps, construction of the extracellular total RNA high-throughput sequencing library as low as 1 ng can be completed within 4 h, the library is high in data quality and almost does not contain rRNA, and multiple types of RNA such as miRNA, lncRNA and mRNA are detected. The invention can provide a simple and effective tool for screening development and disease related RNA biomarkers.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and a kit for constructing an extracellular trace free RNA sequencing library. Background Art

[0002] As a biomarker, extracellular fluid free RNA (exRNA) has better diversity, tissue specificity, and is more sensitive to developmental and disease changes compared to DNA and protein. Compared with puncture to obtain tissue samples, the acquisition of extracellular fluid samples has significant advantages such as being simpler and non-invasive, and has greater potential in clinical diagnosis. At present, some scholars have screened out cfRNA markers for predicting preeclampsia and miscarriage through high-throughput sequencing of plasma free RNA (Circulating Free RNA, cfRNA), and can monitor fetal development progress through cfRNA; some researchers have also screened markers for tumor diagnosis through high-throughput sequencing of exosome RNA. These studies have greatly promoted our monitoring of developmental conditions, prediction and diagnosis of diseases.

[0003] However, the biggest challenge encountered by scholars during the research process is that unlike the abundant RNA in cells or tissues, the exRNA obtained from extracellular fluid is usually trace (usually <10ng), and contains a large amount of rRNA; with such a trace amount of exRNA, the construction of a total RNA library without rRNA usually encounters the problem of library construction failure or poor data quality, and too little useful information is obtained, which cannot be used for subsequent analysis. Although there are currently imported commercial kits (for example: SMARTer Stranded Total RNA-Seq Kit -Pico Input Mammalian, hereinafter referred to as SMART) that can be used to construct exRNA libraries, the target sample designed by the kit is the trace amount of RNA in cells or tissues, which is not completely suitable for constructing libraries of partially degraded exRNA. Moreover, the kit is expensive and the operation steps are relatively cumbersome. The above problems limit the application of exRNA in research such as development, clinical disease prediction and diagnosis. Summary of the invention

[0004] In order to effectively solve the above technical problems, the present invention provides a novel high-throughput sequencing library construction method and kit for trace free RNA in extracellular fluid.

[0005] In a first aspect, the present invention claims a method and a kit for constructing a high-throughput sequencing library of trace amounts of free RNA in extracellular fluid.

[0006] The implementation steps of the exRNA high-throughput sequencing library provided by the present invention are as follows: S1. Based on the source of extracellular fluid, select an appropriate method to extract and purify exRNA.

[0007] S2. Add Mg to the exRNA extract obtained in step S1. 2+ Template replacement reaction buffer was used to fragment RNA at high temperature.

[0008] S3. Antigens for rRNA removal (i.e., Fast rRNA Removal Mix (Novozyme, N460–01)), reverse transcription random primers containing partial adapter sequences (oligo dN), and dNTPs were added simultaneously in step S2, and rRNA removal and reverse transcription primer annealing reactions were performed after RNA fragmentation at high temperature.

[0009] S4. Add template switching reverse transcriptase and template switching oligonucleotide (TSO) to the product obtained in step S3 to reverse transcribe the total RNA except rRNA into cDNA.

[0010] S5. The cDNA obtained in step S4 was amplified by the first round of PCR using primers P5_short / P5_short_extend and P7_short / P7_short_extend, and the PCR product was purified by magnetic beads.

[0011] S6. The first-round PCR product obtained in step S5 is subjected to a second-round PCR using an Illumina adapter containing a barcode, and finally an exRNA high-throughput sequencing library with rRNA removed is obtained.

[0012] The extracellular fluid sample in step S1 includes any one of plasma, exosomes, cerebrospinal fluid, saliva, breast milk, follicular fluid, urine or semen.

[0013] In some embodiments, in the step S1, the obtained sample to be tested (extracellular fluid sample), for example, blood is collected in a blood collection tube containing EDTA, gently inverted and mixed, centrifuged at 4°C, 1900g for 10 min, and the upper plasma is collected. 200 μl of the collected plasma is taken, and plasma exRNA is extracted using miRNeasy Serum / Plasma Advanced Kit according to its instructions, and finally exRNA is eluted from the column with 15 μl of enzyme-free water.

[0014] In some embodiments, specifically, the primer information of the present invention is as follows: Reverse transcription random primer containing partial adapter sequence SEQ ID NO: 1 (oligo dN): 5'-AGACGTGTGCTCTTCCGATCTNNNNNN-3'; Template replacement oligonucleotide SEQ ID NO: 2 (TSO): 5'-CACGACGCTCTTCCGATCTNNNNrGrG+rG-3' (+ indicates locked nucleic acid); The first round of PCR pre-amplification primers SEQ ID NO: 3 (P5_short): 5'-CACGACGCTCTTCCGATCT-3' or P5_short extended to the 5' end by 1-14 bases according to the following underlined sequence SEQ ID NO: 4 (P5_short_extend): 5'- ACACTCTTTCCCTA CACGACGCTCTTCCGATCT-3′; The first round of PCR pre-amplification primer SEQ ID NO: 5 (P7_short): 5'-AGACGTGTGCTCTTCCGATCT-3' or based on P7_short, extend 1-13 bases to the 5' end according to the following underlined sequence PSEQ ID NO: 6 (P7_short_extend): 5'- GTGACTGGAGTTC AGACGTGTGCTCTTCCGATCT-3′; The second round PCR primers are Illumina adapters containing barcodes, namely SEQ ID NO: 7-SEQ ID NO: 26, see Appendix 1.

[0015] Preferably, the reaction system of RNA fragmentation and rRNA blocking in steps S2 and S3 is: exRNA 6μl, FastrRNA Removal Mix (Novozyme, N460-01) 0.5μl, Template Replacement Reaction Buffer (NEB, M0466L) 3μl, Random Primer (oligo dN) containing partial adapter sequence 2μl, dNTP (10mM) 2μl, Enzyme-free water 1.5μl; The reaction procedure is: 90-95℃ 3 min -8 min, 70-75℃ 30s-1 min, 50-55℃ 30s -1 min, 4℃ storage. In some preferred cases, the RNA fragmentation reaction is 94℃ 5 min, 75℃ 1 min, 55℃ 1 min.

[0016] Preferably, the template replacement reverse transcription reaction system described in step S4 is: 15 μl of the reaction product of step S3, 2 μl of template replacement reaction buffer, 1 μl of TSO, and 2 μl of template replacement reverse transcriptase mixture. The reaction procedure is: 42°C for 90 min, 85°C for 5 min, and storage at 4°C. The template replacement reverse transcriptase mixture is purchased from New England Biolabs (NEB, M0466L), and can also be replaced by other reverse transcriptases that can add CCC (three cytosines) to the 3' end of the reverse transcription product.

[0017] Preferably, the reaction conditions of the first round of PCR pre-amplification in step S5 are: the reaction conditions of the first round of PCR amplification are: 98°C 40-45 s; 98°C 10-20 s, 62°C 15-25 s, 72°C 2-4 min, 10-16 cycles; 72°C 5-8 min; 4°C storage. In some preferred cases, 98°C 45 s; 98°C 10 s, 62°C 15 s, 72°C 2 min, 10-16 cycles; 72°C 5 min; 4°C storage. The PCR product is purified using 1.8× DNA purification magnetic beads.

[0018] Preferably, the reaction conditions of the second round of PCR pre-amplification in step S6 are: 98°C 40-45 s; 98°C 10-20 s, 62°C 15-25 s, 72°C 2-4 min, 10-16 cycles; 72°C 5-8 min; 4°C storage. In some preferred cases, 98°C 45 s; 98°C 10 s, 62°C 15 s, 72°C 2 min, 4-12 cycles; 72°C 5 min; 4°C storage. The PCR product is purified using 1× DNA purification magnetic beads.

[0019] In the second aspect, the present invention claims a three-step reaction system and method for exRNA fragmentation, rRNA removal and reverse transcription primer annealing, which comprises steps S2 and S3 in the method described in the first aspect above.

[0020] In a third aspect, the present invention claims a primer set as follows: The reverse transcription primer oligo dN in the method of the first aspect described above, and the first round PCR pre-amplification primers P5_short (also including P5_short_extend) and P7_short (also including P7_short_extend).

[0021] In a fourth aspect, the present invention claims the use of the primers and two rounds of PCR described in steps S5 and S6 of the method shown in the first aspect above in constructing a micro-exRNA high-throughput sequencing library.

[0022] Compared with the prior art, the present invention has the following innovative features and beneficial effects: 1. The present invention only requires a trace amount of exRNA (as low as 1ng) to complete the construction of an exRNA high-throughput sequencing library without rRNA, providing a simple and effective tool for screening exRNA biomarkers related to development, disease prediction and diagnosis.

[0023] 2. The present invention innovatively combines exRNA fragmentation, rRNA removal and reverse transcription primer annealing into one reaction system. The three reactions only take 7 min, and the rRNA removal efficiency is higher (see Figure 2 ). The total time for library construction in the present invention is less than 4 h, which greatly simplifies the operation steps and improves the work efficiency and the success rate of library construction.

[0024] 3. The present invention adopts a two-round PCR amplification library method. In the first round of PCR, shorter primers are used to effectively avoid the problem of primer dimers easily forming during PCR amplification of low starting amount samples, thereby increasing the output of effective library data (see Figure 2 and Figure 3 ).

[0025] 4. The number of plasma cfRNA genes detected by the present invention is about twice that detected by commercially available kits (see Figure 4 ), the detected RNA information is more abundant and comprehensive. This kit does not need to add PolyA tail to short-chain RNA such as miRNA to construct its sequencing library. It can detect short-chain RNA and long-chain RNA including miRNA, as well as non-coding RNA and coding RNA (see Figure 5 ).

[0026] 5. The cost of library construction is significantly reduced, only 20% of commercially available kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of Example 1 of the present invention and the library construction method of SMARTer Stranded Total RNA-Seq Kit (abbreviated as SMART).

[0028] Figure 2 The ratios of rRNA, uniquely mapped and unmapped reads in human plasma samples detected by the SMART kit and the kit of the present invention.

[0029] Figure 3a. The fragment distribution diagram of the exRNA-seq library after the first round of PCR was performed without using primers P5_short and P7_short, and the second round of PCR amplification was performed directly with the adapter primers; b. The fragment distribution diagram of the exRNA-seq library after two rounds of PCR amplification.

[0030] Figure 4 The number of genes with FPKM>1 in human plasma samples detected by the SMART kit and the kit of the present invention.

[0031] Figure 5 The present invention detects the percentages of different types of RNA in human plasma. DETAILED DESCRIPTION

[0032] The present invention will be further explained in detail below with reference to the drawings and specific implementation cases in the specification. It should be pointed out that these implementation cases are only used to illustrate the present invention and do not constitute a limitation on the scope of protection of the present invention. In the following implementation cases, unless otherwise explicitly stated, the experimental means adopted are all conventional methods; the materials and reagents used are all reagents and materials available on the market.

[0033] Example 1 Extracting exRNA from plasma to construct a high-throughput sequencing library 1. Experimental methods (1) The samples to be tested in this example are plasma from two healthy people, and only 200 μl is needed. Take 500 μl of whole blood, collect it in a blood collection tube containing EDTA, gently invert it upside down to mix, centrifuge it at 4°C, 1900g for 10 min, and take the upper plasma.

[0034] (2) Take 200 μl of the plasma collected in step (1) and extract plasma exRNA using the miRNeasy Serum / Plasma Advanced Kit according to its instructions. Finally, elute the exRNA from the column with 15 μl of enzyme-free water.

[0035] (3) Take 6 μl of the exRNA obtained in step (2) for library construction. The schematic diagram of the library construction process is as follows: Figure 1 Prepare the exRNA fragmentation, rRNA removal and reverse transcription primer annealing reaction system according to Table 2 and mix well:

[0036] In Table 2, Fast rRNA Removal Mix (Novuzumab, N460-01); Template Replacement Reaction Buffer (NEB, M0466L). oligo dN: SEQ ID NO: 1.

[0037] The temperature programs for the three reactions are shown in Table 3:

[0038] (4) Prepare the template replacement reverse transcription reaction system according to Table 4 below:

[0039] After mixing the above reaction system, incubate at 42°C for 90 min, 85°C for 5 min, and store at 4°C. TSO: SEQ ID NO: 2.

[0040] (5) Prepare the first round of PCR reaction system according to Table 5 below:

[0041] P5_short: SEQ ID NO:3; P7_short: SEQ ID NO:5.

[0042] After the first round of PCR reaction system is mixed, the reaction is carried out according to the temperature program in Table 6 below:

[0043] The first-round PCR products were purified using 1.8 × DNA purification magnetic beads and eluted with 25 μl enzyme-free water.

[0044] (6) Prepare the second round of PCR reaction system according to Table 7 below:

[0045] DM501: SEQ ID NO: 7; DM702: SEQ ID NO: 16.

[0046] After the second round of PCR reaction system is mixed, the reaction is carried out according to the temperature program in Table 8 below:

[0047] The second-round PCR products were purified using 1× DNA purification magnetic beads and eluted with 20 μl enzyme-free water to finally obtain the plasma exRNA high-throughput sequencing library.

[0048] 2. Experimental Results (1) With SMART kit (using Figure 1 Compared with the construction model diagram shown in the figure, the kit of the present invention adopts a new rRNA removal method, and the total library construction time is shortened by 78 min; another difference is that the first round of PCR of the present invention adopts short-chain primers instead of full-length Illumina adapters, which can effectively avoid the formation of primer dimers.

[0049] (2) From Figure 2It can be seen that the residual amount of rRNA in the library from which rRNA was removed by the SMART kit was still >8%, and the reads mapped to the genome accounted for about 50%; while the residual amount of rRNA in the library from which rRNA was removed by the present invention was <3%, and the reads mapped to the genome were >75%, and the rRNA in the library from which rRNA was not removed accounted for about 40%.

[0050] (3) Figure 3 The size distribution of DNA fragments in the exRNA library was checked for Qsep 400. Figure 3 a is an exRNA-seq library that was amplified by PCR directly using the second-round adapter primers without using primers P5_short and P7_short for the first round of PCR (i.e., the method and steps are the same as in Example 1, except that step (5) is not performed). The main peak is located at around 150 bp, which is the primer dimer formed by the adapter primers (the peak marked by the blue dotted box), and the RNA fragment to be tested cannot be effectively amplified. Figure 3 b is the exRNA-seq library subjected to two rounds of PCR in Example 1, which is mainly distributed between 180-600 bp, which is a normal library fragment distribution.

[0051] (4) From Figure 4 It can be seen that the number of genes with plasma cfRNA FPKM>1 detected by the SMART kit is about 10,000, and the number of genes with plasma cfRNA FPKM>1 detected by the present invention is about 20,000.

[0052] (5) Figure 5 FeatureCounts was used to calculate the expression level of each gene in exRNA in plasma, and then R language was used to count each type of RNA and its percentage. The results showed that mRNA was the most abundant, accounting for 35.2%, followed by Mt_rRNA (15.7%), lncRNA (15.0%), processed_pseudogene (12.4%), misc_RNA (7.8%), and non-coding RNA accounted for more than half.

[0053] Appendix 1: Second round PCR primers (s stands for thiolation, bold is the index sequence) .

Claims

1. A method for constructing a sequencing library of extracellular trace free RNA, characterized in that: The following steps are involved: S1. Extraction and purification of exRNA from extracellular fluid; S2. In the presence of Mg 2+ The exRNA was fragmented at high temperature in the template replacement reaction buffer; S3. While S2 is being performed, rRNA removal reaction and reverse transcription primer annealing reaction are performed; S4. Adding a template replacement reverse transcriptase and a template replacement oligonucleotide to the product obtained in step S3 to reverse transcribe the total RNA except rRNA into cDNA; S5. The cDNA obtained in step S4 is subjected to the first round of PCR amplification using primers P5 and P7, and the PCR product is purified by magnetic beads to obtain the first round of PCR product; S6. The first-round PCR product obtained in step S5 is subjected to a second-round PCR using an Illumina adapter containing a barcode, and finally an exRNA high-throughput sequencing library is obtained.

2. The method according to claim 1, characterized in that: The extracellular fluid sample in step S1 includes any one of plasma, exosomes, cerebrospinal fluid, saliva, breast milk, follicular fluid, urine or semen.

3. The method according to claim 1, characterized in that In steps S2 and S3, a removal agent is added to the rRNA removal reaction, and the removal agent is selected from Fast rRNA Removal Mix N460-01; The reverse transcription primer is a random primer SEQ ID NO: 1 containing a partial linker sequence.

4. The method according to claim 3, characterized in that In steps S2 and S3, the reaction procedures for exRNA fragmentation, rRNA removal and reverse transcription primer annealing were: 90-95 °C for 3 min-8 min, 70-75 °C for 30s-1 min, 50-55 °C for 30s-1 min, and stored at 4 °C.

5. The method according to claim 1, characterized in that In step S4, the template replacement oligonucleotide sequence is SEQ ID NO:

2.

6. The method according to claim 1, characterized in that In step S5, the sequences of primer P5 are SEQ ID NO: 3 and SEQ ID NO: 4; The sequences of the primers P7 are SEQ ID NO:5 and SEQ ID NO:

6.

7. The method according to claim 6, characterized in that In step S5, the reaction conditions for the first round of PCR amplification are: 98°C 40-45 s; 98°C 10-20 s, 62°C 15-25 s, 72°C 2-4 min, 10-16 cycles; 72°C 5-8 min; and stored at 4°C.

8. The method according to claim 1, characterized in that In step S5, the primers for the second round of PCR amplification in step S7 contain a barcode, including any one of SEQ ID NO:7-SEQ ID NO:26, and the reaction conditions are: 98°C 40-45s; 98°C 10-20s, 62°C 15-25s, 72°C 2-4 min, 10-16 cycles; 72°C 5-8 min; and stored at 4°C.

9. A kit comprising an extracellular trace free RNA sequencing library constructed by the method described in any one of claims 1 to 8.

10. Use of the kit according to claim 9 for detecting total RNA, wherein the total RNA includes short-chain RNA including miRNA and long-chain RNA, non-coding RNA and coding RNA.

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