WO2016127345A1 - Method for constructing and sequencing small rna library - Google Patents

Abstract

Provided is a method for constructing a small RNA library, wherein the constructed small RNA library is suitable for second-generation sequencing technology.

Description

Library construction and sequencing method of small RNA Technical field

The present invention relates to the field of cancer diagnosis. More specifically, the invention relates to libraries of cancer-associated small RNAs.

Background technique

In recent years, the global cancer incidence situation is severe, and the morbidity and mortality have continued to rise. According to the World Cancer Report 2014 published by the International Agency for Research on Cancer, the number of cancers worldwide has risen from 12.7 million in 2008 to 14.1 million in 2012, and is expected to reach 22 million per year in the next 20 years. The death toll will also rise from 8.2 million per year to 13 million. In China, the incidence of cancer in 2012 accounted for almost half of the world's cancer, ranking first. As early as the National Cancer Center published in the 2012 Cancer Registration Annual Report, there are about 3.12 million new cancer cases in China each year, and 6 people are diagnosed as malignant tumors every minute. About 2.7 million cancer deaths occur every year in the country. Five people died of cancer. At present, the probability of death of cancer in China is 13% [1, 2], and cancer poses a great threat to human health and life.

Since the 20th century, modern medical technology has made great progress in the diagnosis and treatment of cancer, but there are still problems such as untimely diagnosis and poor therapeutic effects. Clinical studies have shown that the cure rate of carcinoma in situ is close to 100%, the 6-year survival rate of patients with stage I lung cancer is 60%-90%, and the 5-year survival rate of patients with stage IIIb and IV is only 5%-20%, indicating cancer. Early diagnosis is the key to improving prognosis. However, due to the lack of ideal early diagnosis techniques, the early diagnosis rate of lung cancer is only about 14% [3]. Therefore, there is an urgent need to develop early cancer diagnosis techniques to improve patient survival. In addition, effective treatment of cancer can also greatly reduce Cancer mortality. However, in the cure rate of cancer, developed countries have reached 65%, while China has only about 25%, and the current clinical treatment of cancer is mainly surgical resection and radiotherapy. When radiotherapy and chemotherapy kill cancer cells, they also cause serious damage to normal human cells. Therefore, it is necessary to develop new and effective treatment technologies. In recent years, with the deepening of research on cancer, biological treatment and targeted therapy of tumors have become more and more important in cancer treatment by virtue of their non-invasive or minimally invasive properties, and become the main target of cancer treatment. direction.

Small RNA is an important class of in vivo regulatory molecules, including miRNA, piRNA and siRNA. Small RNA works through a variety of pathways, including mRNA degradation, translational inhibition, heterochromatin formation, and DNA removal to regulate growth and development of organisms, and even plays a key role in the development of diseases such as cancer. The expression of Small RNA is quite different between cancer tissues and non-cancerous tissues, and specific expression of small RNA is associated with specific cancers. Therefore, small RNA can be used as a molecular marker for diagnosing cancer, such as derived from exosomes. (exosome) miR-21 can be used as a prognostic indicator for several cancers, and miR-21 is associated with miR-31 and esophageal cancer. In addition, if the expression and release of miRNAs in the exosomes can be controlled or the exosomes are carried to deliver molecules such as therapeutic miRNAs to target cells, targeted therapy for some cancers and diseases can be performed. By constructing a small RNA library and performing large-scale sequencing analysis of the small RNA library, the genome-wide small RNA map of the species can be obtained, including the mining of new small RNA molecules, the prediction and identification of the target genes, and the differences between samples. Expression analysis, scientific applications such as Small RNA clustering and expression profiling.

With the development of second-generation sequencing technology, small RNA libraries constructed using different library construction techniques are applicable to different sequencing platforms, such as the Solexa sequencing platform and the Ionproton sequencing platform. Complete Genomics (CG) sequencing is a new second-generation sequencing technology that uses high-density DNA nanochip technology to embed DNA nanospheres on a chip and anchor with non-continuous, non-chained probes (cPAL). Technology read sequences with higher throughput and low cost per genome sequencing To $1,000. In summary, the CG library construction sequencing technology has the characteristics of low cost of construction, simple operation, short time, high success rate of database construction, fast sequencing, etc., and has high promotion value.

Summary of the invention

The invention describes a novel method for building small RNA, which can be used for library construction of small RNA from all sources, and the constructed small RNA CG library is suitable for second generation sequencing technology Complete Genomics (CG) sequencing. platform.

In a first aspect, the invention provides a method of constructing a small RNA CG library, the method comprising the steps of:

1. Extracting the small RNA fragment of the sample;

2. The small RNA fragment is ligated to the 3' linker of the CG library and the 5' linker of the CG library;

3. The small RNA ligated to the 3' and 5' linkers of the CG library is reverse transcribed into cDNA;

4. PCR amplification of reverse transcription products;

5. Cyclization of the single-stranded DNA of the PCR amplification product and recovery of the cyclized product;

6. Library quality control and concentration determination.

In one embodiment, the small RNA CG library is a cancer small RNA CG library, the sample is a cancer sample.

In one embodiment,

3' linker: 5'-GTCTCCAGTCGAAGCCCGATCxxxxxxxxxxGAGCTTGTC T-3' (SEQ ID NO. 1) (where xxxxxxxxxx represents a 10 bp Barcode sequence, ie, a sequence for distinguishing different samples when multiple samples are mixed and sequenced)

5' linker: 5'-UCCUAAGACCGCUUGGCCUCCGACUU-3' (SEQ ID NO. 2).

In one embodiment, the reverse transcription primer RT-primer: 5'-AGACAAGCTCxxxxxxxxxxGATCGGGCTTCG ACTGGA GAC-3' (SEQ ID NO. 3) (where xxxxxxxxxx represents a 10 bp Barcode sequence).

In one embodiment, PCR amplification of the reverse transcription product uses primer ON0639: 5'-TCCTAAGACCGCTTGGCCTCCGACTT-3' (SEQ ID NO. 4).

In one embodiment, cyclization of single-stranded DNA uses ON1587: 5'-TCGAGCTTGTCTTCCTAAGACCGC-3' (SEQ ID NO. 5).

In one embodiment, SEQ ID NO. 1 is selected from the group consisting of SEQ ID NO. 6-9. In one embodiment, SEQ ID NO. 3 is selected from the group consisting of SEQ ID NO. 10-13. Wherein SEQ ID NO. 6-9 corresponds to SEQ ID NO. 10-13.

In a second aspect, the invention provides a method for sequencing a small RNA CG library, the method comprising the steps of:

The small RNA CG library constructed in accordance with the first aspect of the invention was sequenced using a CG sequencing platform.

In one embodiment, the small RNA CG library is a cancer small RNA CG library.

The small RNA library construction method adopted by the invention is applicable to the complete genome (Complete Genomics, CG) sequencing platform, and has high sequencing throughput, low cost of construction, simple operation, short time, high success rate of database construction and fast sequencing. The library sequencing method has a high promotion value, and can quickly and efficiently construct a small RNA library from the human body and subsequent sequencing analysis for early diagnosis of cancer and development of subsequent personalized targeted therapy.

The small RNA library constructed by the method of the invention adopts the CG 1adapter construction method, and has the characteristics of low construction cost, simple operation, short time, high success rate of database construction and fast sequencing.

DRAWINGS

Figure 1. Flow chart of the library construction of this experiment.

detailed description

The invention provides a novel method for building and sequencing small RNA, which can be used for constructing a small RNA library of all sources, and the constructed small RNA CG library is suitable for the second generation sequencing technology complete genomics (CG). Sequencing platform.

The construction of the DNA CG library already has a mature database construction scheme, and the construction of the small RNA CG library of the present invention is a relatively new library construction method, and the difference from the DNA CG library construction is mainly manifested in the following aspect:

1) The construction of the DNA CG library requires dephosphorylation and end-filling of the library fragments, but the construction of the small RNA CG library of the present invention does not require this step.

2) The construction of the DNA CG library in which the 3' and 5' linkers are in the same reaction system and are ligated to both ends of the library fragment, while the 3' and 5' junctions of the small RNA library of the present invention are Two reaction systems are respectively connected to the two ends of the library fragment, and the 3' and 5' junctions are independently designed and synthesized by the University of China for small RNA.

3. Construction of the DNA CG library Immediately after the ligation of the linker, the nick translation reaction is performed and then PCR amplification is performed. The construction of the small RNA CG library does not require the nick translation reaction, and only the library fragment ligated to the linker is inverted. PCR amplification after recording,

4. Construction of the DNA CG library can be performed after single-strand separation after PCR amplification, and the construction of the small RNA CG library needs to be purified by non-denaturing PAGE gel to recover the PCR amplification product and then subjected to single-strand separation treatment.

In a specific embodiment, the method of the present invention mainly includes the following two aspects:

(1) Construction of small RNA CG library

1. The small RNA fragment is ligated to the 3' linker of the CG library,

2. The small RNA fragment is ligated to the 5' linker of the CG library,

3. The small RNA ligated to the 3' and 5' linkers of the CG library is reverse transcribed into cDNA.

4. PCR amplification of reverse transcription products,

5. Purification of the PCR amplification product by non-denaturing PAGE gel (for example, 6% PAGE gel).

6.Qubit quantitative purification of the recovered PCR product,

7. Single-strand separation of the PCR product after purification and recovery,

8. Cyclization of single-stranded DNA,

9. Digestion of uncircularized single-stranded DNA,

10. Purification of the digested product (eg using PEG32beads),

11. Library quality control and concentration determination;

(2) Small RNA CG library sequencing and data analysis under the machine,

1.Small RNA CG library CG sequencing platform on the machine sequencing,

2. Analysis of the data of the small RNA CG library.

In one embodiment,

3' linker: 5'-GTCTCCAGTCGAAGCCCGATCxxxxxxxxxxGAGCTTGTC T-3' (SEQ ID NO. 1) (where xxxxxxxxxx represents a 10 bp Barcode sequence)

5' linker: 5'-UCCUAAGACCGCUUGGCCUCCGACUU-3' (SEQ ID NO. 2).

In one embodiment, the reverse transcription primer RT-primer: 5'-AGACAAGCTCxxxxxxxxxxGATCGGGCTTCG ACTGGA GAC-3' (SEQ ID NO. 3) (where xxxxxxxxxx represents a 10 bp Barcode sequence).

In one embodiment, PCR amplification of the reverse transcription product uses primer ON0639: 5'-TCCTAAGACCGCTTGGCCTCCGACTT-3' (SEQ ID NO. 4).

In one embodiment, the cyclization of single-stranded DNA uses ON1587: 5'-TCGAGCTTGTCTTCCTAAGACCGC-3' (SEQ ID NO. 5).

Example

In this example, the initial sample of the library was the small RNA in the exosomes derived from the serum of 4 lung cancer patients. Concentration of the first use of the SBI's ExoQuick ^TM Exosomes precipitation Solution outer enriched in the serum, and then use the outer small RNA extracted SearMir kit SBI's body, small RNA was extracted 0.162-0.424ng / Ul, the volume is 30ul.

The specific sequences of the RNA 3' linker, RNA 5' linker, RT-primer, ON0639, and ON1587 used in this example are as follows:

1.3' linker: 5'-GTCTCCAGTCGAAGCCCGATCxxxxxxxxxxGAGCTTGTC T-3' (SEQ ID NO. 1) (where xxxxxxxxxx represents a 10 bp Barcode sequence, ie, a sequence used to distinguish different samples when multiple samples are mixed and sequenced), used in this example The 4's 3' joints with 10bp Barcode are independently designed and synthesized by Huada. The sequences are:

5'-GTCTCCAGTCGAAGCCCGATCATAAGGCAGTGAGCTTGTC T-3' (SEQ ID NO. 6)

5'-GTCTCCAGTCGAAGCCCGATCTTGATAGATTGAGCTTGTC T-3' (SEQ ID NO. 7)

5'-GTCTCCAGTCGAAGCCCGATCCCTTCCTGGTGAGCTTGTC T-3' (SEQ ID NO. 8)

5'-GTCTCCAGTCGAAGCCCGATCAATATCTCTCGAGCTTGTC T-3' (SEQ ID NO. 9)

2.5' linker: 5'-UCCUAAGACCGCUUGGCCUCCGACUU-3' (SEQ ID NO. 2)

3. RT-primer: 5'-AGACAAGCTCxxxxxxxxxxGATCGGGCTTCG ACTGGA GAC-3' (SEQ ID NO. 3) (where xxxxxxxxxx represents a 10 bp Barcode sequence)

In this embodiment, four RT-primers with 10 bp Barcode are used by Huada to design and synthesize them. The sequences are:

5'-AGACAAGCTCACTGCCTTATGATCGGGCTTCGACTGGAGAC-3' (SEQ ID NO. 10)

5'-AGACAAGCTCAATCTATCAAGATCGGGCTTCGACTGGAGAC-3' (SEQ ID NO. 11)

5'-AGACAAGCTCACCAGGAAGGGATCGGGCTTCGACTGGAGAC-3' (SEQ ID NO. 12)

5'-AGACAAGCTCGAGAGATATTGATCGGGCTTCGACTGGAGAC-3' (SEQ ID NO. 13)

4. ON0639: 5'-TCCTAAGACCGCTTGGCCTCCGACTT-3' (SEQ ID NO. 4)

5. ON1587: 5'-TCGAGCTTGTCTTCCTAAGACCGC-3' (SEQ ID NO. 5)

The steps of the specific small RNA CG database construction in this embodiment are as follows:

1. The small RNA fragment is ligated to the 3' linker of the CG library

(1) Reaction volume and conditions

Reagent	Volume (ul)
		Small RNA (nuclease-free)	5
10uM RNA 3' connector	1
		total	6

Placed on ice at 70 ° C for 2 min in Thermal cycler

(2) Mix configuration and reaction conditions

Add the configured Mix to the reaction product of (1), mix it, and react at 25 ° C for 2 hours in the Thermal cycler, then keep it at 12 ° C.

(3) 3' joint annealing

Add 0.5ul of 100uM RT-primer to the reaction product of (2), mix, then react at 75 ° C for 5 min in the Thermal cycler, react for 30 min at 37 ° C, and react for 15 min at 25 ° C.

2. The small RNA fragment is ligated to the 5' linker of the CG library.

(1) Take a new RNase-free PCR tube, add 1 ul of 10 uM RNA 5' linker, react at 70 ° C for 2 min in Thermal cycler, and immediately place on ice.

(2) Mix configuration and reaction conditions

Reagent	volume
		10mM ATP	1
10U/ul T4 RNA ligase 1 (BioLabs)	1
		40U/ul RNase inhibitor	0.5
total	2.5

The prepared Mix was added to the reaction product of the above (1) 5' linker, mixed, and then the mixture was added to the 3' joint annealing product of the above 1 (3), and reacted at 20 ° C for 1 hour in a Thermal cycler.

3. Reverse transcription of cDNA from the 3' and 5' linkers of the CG library into cDNA

(1) Reverse transcription system and reaction conditions

Add 10.5 ul of the system to the reaction product of the second step, mix, react at 42 ° C for 40 min in the Thermal cycler, react for 15 min at 70 ° C, then keep at 12 ° C

4. PCR amplification of reverse transcription products

(1) PCR reaction system

Add the configured 25ul system to the reverse transcription product of the above 3, a total of 50ul system

(2) PCR reaction conditions

Purification of PCR amplification products by 5.6% non-denaturing PAGE gel

(1) 2 ul of 20 bp DNA ladder, 10 ul of 6x loading buffer in the PCR product, 180V constant pressure electrophoresis

(2) Making a cannula: use a fired syringe needle to puncture a small hole in the bottom of a 500 ul EP tube, then place it in a 2 ml EP tube, and wrap the lid of the 2 ml tube with the cling film and the lid to the tube body.

(3) The cut target PAGE gel was placed in a 500 ul EP tube with small holes, centrifuged at 12000 rpm for 2 min at room temperature, and the PAGE block was broken.

(4) Add 0.3M NaCl to the broken PAGE block, then heat the mixer to 350 rpm, 25 ° C, 2 hours.

(5) Transfer the mixed mixture to a spin-x 0.45um filter column, centrifuge at 5 °C for 5 min at 13000 rpm.

(6) 1/10 volume of 3 M sodium acetate, 2 ul of 5 mg/ml glycogen, 2-3 volumes of absolute ethanol, and then precipitated at -80 ° C for 1 hour in the filtrate.

(7) Centrifuge at 13000 rpm for 30 min at 4 ° C, then add 1 ml of 80% ethanol to gently bake off the pellet for washing.

(8) Centrifuge at 13000 rpm for 5 min at 4 ° C, dry at room temperature, add 22 ul elution buffer to dissolve the precipitate.

6.Qubit quantitative purification of recovered PCR products

The purified PCR product was subjected to concentration determination using a Qubit dsDNA HS assay kit.

7. Single-strand separation of PCR products after purification and recovery

(1) Add 1xTE of the DNA sample dissolved in the above elution buffer to a total volume of 60ul

(2) Prepare the following reagents in advance: Dilute 100% Tween 20 to 0.5% Tween 20, and mix with Streptavidin Beads

(3) 1x BBB (Bead Binding Buffer, 110 mM Tris-HCl, 200 mM NaCl) / 0.5% Tween 20 Mix, 1 x BWB (Bead Wash Buffer, 10 mM Tris-HCl, 40 mM NaCl) / 0.5% Tween 20 Mix, 0.1 M NaOH, 15 min in advance. The configuration method is as follows:

a.1X BBB/0.5%Tween20Mix

Reagent	Volume (ul)
		1X BBB	30
0.5% Tween20	0.3
		total	30.3

b.1X BWB/0.5%Tween20Mix

Reagent	Volume (ul)
		1X BWB	2000
0.5% Tween20	20
		total	2020

c.0.1M NaOH

Reagent	Volume (ul)
		0.5M NaOH	5.2
water	20.8
		total	26

(4) Wash Streptavidin Beads (connected with biotin B) as follows

a. Take 30ul of Streptavidin Beads into each 1.5ml EP tube, then add 4 times the volume of 1XBBB, mix and place on a magnetic stand to absorb statically, adjust the direction of the EP tube, so that The beads are swam back and forth in the 1XBBB lotion (rotate the EP tube twice), and after discarding the supernatant, repeat the above operation once.

b. Remove the non-stick tube and add 1 volume (30 ul) of 1X BBB/Tween Mix suspension, mix and let stand at room temperature.

(5) Add 20 ul of 4XBBB to 60 ul of PCR product mixture, then transfer to a non-stick tube containing 30 ul of 1X BBB/0.5% Tween 20 Mix dissolved beads. Mix the mixture at room temperature for 15-20 min at room temperature. Lightly play once

(6) Place the above non-stick magnetic frame for 3-5 minutes, discard the supernatant, and wash it twice with 1 ml of 1XBWB/0.5% Tween20Mix. The method is the same as the washing method of Streptavidin Beads.

(7) Add 26ul of 0.1M NaOH to the above beads, mix by pipetting for 10min, then place on the magnetic stand for 3-5min, and take the supernatant to the new PCR tube.

(8) Add 13ul of 0.3M MOPS to the above PCR tube, mix and set aside, the product of this step can be frozen at -20 °C.

8. Cyclization of single-stranded DNA

(1) Add 10ul of 20uM ON1587 to the 39ul sample obtained in the previous step.

(2) Mix configuration and reaction conditions

Reagent	Volume (ul)
		water	4.2
10x TA Buffer(LK1)	6
		100mM ATP	0.6
600 U/ul ligase	0.2
		total	11

Mix the Mix well and centrifuge, then add to the mixture of (1), shake for 10 s, and mix after transient centrifugation.

37 ° C reaction for 1.5 hours

9. Digestion of uncircularized single-stranded DNA

(1) Mix configuration and reaction conditions

Reagent	Volume (ul)
		10x TA Buffer(LK1)	1
20U/ul ExoI	3
		200U/ul ExoIII	1
total	5

Mix the Mix well and centrifuge, then add to the cyclized product in the previous step, mix for 10 s, and centrifuge at 37 ° C for 30 min.

(2) After the enzyme was cut for 30 min, 2.5 ul of 500 mM EDTA was added to the sample to terminate the enzyme reaction.

10. Purification of the digested product by PEG32beads

(1) Transfer the above-mentioned digestion reaction product to a new 1.5 ml non-stick tube, add 84.5 ul of PEG32beads/0.5% Tween20 (PEG32beads: 0.5% Tween20=100:1), combine at room temperature for 15 min, and mix and mix once.

(2) After the non-stick tube is placed on the magnetic stand for 3-5min, the supernatant is discarded.

(3) Wash 700ul 75% ethanol twice, and wash the non-stick tube in the direction of the front and back, so that the beads move in the ethanol, swimming 2-3 times per wash.

(4) Dry at room temperature, add 27ul TE/0.5% Tween20 (TE: 0.5% Tween20=500:1), combine at room temperature for 15min, mix once in the middle.

(5) Transfer the supernatant to a new 1.5 ml EP tube, and finally obtain the product as a CG library.

11. Library quality control and concentration determination

(1) The library was quantified by Qubit ^TM ssDNA Assay Kit. The ratio of Buffer to dye was 199:1. After mixing, votex and centrifuged for mixing. Take two 190 ul of diluted dye working solution and add 10 ul of two standard votex and centrifuge. Mix and reserve, take 198ul diluted dye working solution to add 2ul sample, votex and centrifuge for Qubit instrument quantification

(II) Small RNA CG library sequencing and data analysis

1.small RNA CG library CG sequencing platform on-machine sequencing

During the library construction process, the 3' junctions of the small RNAs in the serum-derived exosomes of 4 lung cancer patients contained different Barcodes, so the four different sources of small RNA PCR can be expanded in the PAGE gel recovery step. The product is pooled together for recovery and finally a CG hybrid library with 4 different initial samples pooled together. The concentration of the mixed CG 1adapter library was greater than 7.5 fmol/ul and the volume was 20 ul, which met the concentration requirement for sequencing on the CG library (120 fmol required for CG library 1 sequencing sequencing make DNB). The mixed library was sequenced using CG 1adapter 12+19 double-end sequencing to measure 2 lanes (7.5 G/lane).

2. Analysis of the data of the small RNA CG library.

The CG hybrid library was sequenced and analyzed. The results showed that the data of the small RNA CG library in the serum-derived exosomes of 4 lung cancer patients in the mixed library were all larger than 60M reads. The data was found to be 55. A known small RNA containing 36 cancer-associated small RNAs. The results indicate that the small RNA derived from the serum exosome can be constructed into a CG library by using the CG 1adapter construction method with low cost, simple operation, short time and high success rate. The successful construction of the CG library is suitable for high sequencing throughput. , Complete sequencing of the complete genome (Complete Genomics, CG) sequencing platform. By analyzing the data of the Small RNACG library under the database sequencing method, some information about cancer-related small RNA can be obtained, which can lay a foundation for the early diagnosis of cancer and the development of subsequent personalized targeted therapy.

Claims (10)

1. A method of constructing a small RNA CG library, the method comprising the steps of:

   (1) extracting a small RNA fragment of the sample;

   (2) The small RNA fragment is ligated to the 3' linker of the CG library and the 5' linker of the CG library;

   (3) Small RNA ligated to the 3' and 5' linkers of the CG library is reverse transcribed into cDNA;

   (4) PCR amplification of reverse transcription products;

   (5) cyclization of the PCR-amplified product single-stranded DNA and recovery of the cyclized product;

   (6) Library quality control and concentration determination.
2. The method of claim 1, wherein the small RNA CG library is a cancer small RNA CG library, the sample is a cancer sample, and the cancer is preferably lung cancer.
3. The method of claim 1 or 2, wherein the 3' linker is SEQ ID NO. 1: 5'-GTCTCCAGTCGAAGCCCGATCxxxxxxxxxxGAGCTTGTC T-3', wherein xxxxxxxxxx represents a 10 bp Barcode sequence, and/or the 5' linker is SEQ ID NO. 2:5'-UCCUAAGACCGCUUGGCCUCCGACUU-3'.
4. The method of claim 3, wherein SEQ ID NO. 1 is selected from the group consisting of SEQ ID NO. 6-9.
5. The method of any one of claims 1 to 3, wherein the reverse transcription primer RT-primer is SEQ ID NO. 3: 5'-AGACAAGCTCxxxxxxxxxxGATCGGGCTTCG ACTGGA GAC-3' (where xxxxxxxxxx represents a 10 bp Barcode sequence).
6. The method of claim 5, wherein SEQ ID NO. 3 is selected from the group consisting of SEQ ID NO. 10-13.
7. The method of any one of claims 1 to 6, wherein the PCR amplification of the reverse transcription product uses primer SEQ ID NO. 4: 5'-TCCTAAGACCGCTTGGCCTCCGACTT-3'.
8. The method according to any one of claims 1 to 7, wherein the cyclization of the single-stranded DNA uses SEQ ID NO. 5: 5'-TCGAGCTTGTCTTCCTAAGACCGC-3'.
9. A method for sequencing a small RNA CG library, the method comprising the steps of:

   The small RNA CG library constructed according to any one of claims 1-8 was sequenced using a CG sequencing platform.
10. The method of claim 9, wherein the small RNA CG library is a cancer small RNA CG library.

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