METHODS AND KITS OF CONSTRUCTING SEQUENCE LIBRARY FOR USE IN DETECTING CHROMOSOME COPY NUMBER VARIATION

Abstract

Provided herein is a method of constructing a high throughput sequencing library for use in detecting chromosome copy number variation comprising mainly the following steps: (1) subjecting DNAs to be tested to random fragmentation by a double-strand DNA fragmentation enzyme; (2) end-filling and adding poly-adenine at the 3end of the fragmented DNAs; (3) connecting the end-filled DNAs having a 3end poly-adenine with sequencing linkers to obtain connected products; (4) purifying the connected products to obtain the sequencing library; wherein steps (1)-(3) are performed in a single reaction tube. Also provided is a kit for constructing a sequencing library for use in detecting chromosome copy number variation.

Claims

1. A method of rapidly constructing a high throughput sequencing library for use in detecting chromosome copy number variation, characterized in that, the method comprises the following steps: (1) subjecting DNAs to be tested to random fragmentation by a double-strand DNA fragmentation enzyme; (2) end-filling and adding poly-adenine at the 3 end of the fragmented DNAs; (3) connecting the end-filled DNAs having a 3 end poly-adenine with sequencing linkers to obtain connected products; (4) purifying the connected products to obtain the sequencing library; wherein steps (1)-(3) are performed in a single reaction tube.

2. The method according to claim 1, characterized in that, there is no DNA purification steps among steps (1)-(3).

3. The method according to claim 1, characterized in that, the method does not comprise a step of PCR amplification.

4. The method according to claim 1, characterized in that, there is a step of inactivating the double-strand DNA fragmentation enzymes between the step (1) and step (2).

5. The method according to claim 1, characterized in that, the double-strand DNA fragmentation enzyme is an enzyme mixture of a nonspecific notch nuclease and a T7 endonuclease mutant.

6. The method according to claim 4, characterized in that, the nonspecific notch nuclease is a Vvn nuclease, and the T7 endonuclease mutant is a T7 endonuclease having a mutation or mutations in the bridging region of two catalytic domains.

7. The method according to claim 1, characterized in that, step (2) is carried out by T4 DNA polymerase and Taq enzyme.

8. The method according to claim 1, characterized in that, the sequencing linkers are double-strand sequencing linkers matched to sequencing platforms.

9. The method according to claim 1, characterized in that, step (3) is carried out by T4 DNA ligase.

10. The method according to claim 1, characterized in that, the sequencing library is suitable for the second generation high throughput sequencing platforms.

11. A kit for constructing a sequencing library for use in detecting chromosome copy number variation, characterized in that, the kit comprises: double-strand DNA fragmentation enzymes and buffer I for DNA fragmentation, enzymes for DNA end-filling, enzymes for adding poly-adenine at 3 end, sequencing linkers, ligase and buffer III for ligation, wherein the DNA fragmentation, DNA end-filling and addition of poly-adenine at 3 end are carried out in a single reaction tube.

12. The kit according to claim 11, characterized in that, the double-strand DNA fragmentation enzyme is an enzyme mixture of a nonspecific notch nuclease and a T7 endonuclease mutant.

13. The kit according to claim 12, characterized in that, the nonspecific notch nuclease is a Vvn nuclease, and the T7 endonuclease mutant is a T7 endonuclease having a mutation or mutations in the bridging region of two catalytic domains.

14. The kit according to claim 11, characterized in that, the enzyme for DNA end-filling is T4 DNA polymerase, and the enzyme for adding poly-adenine at 3 end is Taq enzyme.

15. The kit according to claim 11, characterized in that, the sequencing linkers are double-strand sequencing linkers matched to sequencing platforms.

16. The kit according to claim 11, characterized in that, the ligase is T4 DNA ligase.

17. The kit according to claim 11, characterized in that, it further comprises buffer II for inactivating the double-strand DNA fragmentation enzymes.

Description

FIGURES

[0026] FIG. 1 is a diagram illustrating the method for constructing a library according to the present invention.

[0027] FIG. 2 shows the sequencing results of a DNA sample to be tested (A) and a control normal sample (B) using libraries constructed according to the method of the present invention.

[0028] FIG. 3 shows the sequencing results of a second DNA sample to be tested (A) and a control normal sample (B) using libraries constructed according to the method of the present invention.

EXAMPLES

Example 1: Library Construction According to the Present Invention and its Use in DNA Sequencing

[0029] This example detected the chromosome aneuploidy abnormality exemplified by Patau syndrome (T13) and chromosome microdeletion exemplified by chromosome 22q11.2 microdeletion syndrome, using the method and kit for constructing a sequencing library for use in detecting chromosome copy number variation according to the present invention. Specifically, the Patau syndrome is a common trisomy syndrome, and is due to the presence of a third copy of chromosome 13. The chromosome 22q11.2 microdeletion syndrome is a clinical syndrome resulted from 22q11.21-q11.23 microdeletion in chromosome 22, and it is one of the most common microdeletion syndromes.

[0030] The sample DNAs are detected according to the following method.

[0031] 1. determining concentration: the concentration of DNA samples to be tested was measured by Qubit florometer, and the initial amounts of DNA samples were adjusted preferably as 10-260 ng.

[0032] 2. constructing a library: a sequencing library was constructed according to the method of the present application, which specifically comprises the following steps:

[0033] (1) preparing a reaction mixture on ice using DNA samples to be tested and a negative control (a normal DNA sample) as indicated in Table 1.

TABLE-US-00001 TABLE 1 Reagents Amount DNA sample 10 ng Double-stranded DNA 1 l fragmentation enzyme Buffer I 1 l EB buffer add to 10 l

[0034] After thorough mixing, the reaction mixture is incubated at 37 C. for 10 min.

[0035] (2) The reaction mixture was transferred onto ice, added with 7 l buffer II, and incubated for 10 min at 65 C. after mixing thoroughly, so as to inactive the double-stranded DNA fragmentation enzyme.

[0036] (3) The reaction mixture was transferred onto ice, added with 1.5 l T4 DNA polymerase and 1.5 l Taq enzyme, and then added to 50 l using the EB buffer. After thorough mixing, the reaction mixture was centrifuged for 5 s at room temperature instantly, and bubbles were removed by slight flicks. Then, the reaction mixture was placed in a normal PCR machine (or a thermo metal bath) to carry out the following incubation steps: 20 min at 37 C., followed by 20 min at 72 C., and finally 5 min at 4 C.

[0037] (4) After incubation in the PCR machine (or a thermo metal bath), the reaction mixture was took out and placed on ice. Meanwhile, a pre-mixture is prepared according to Table 2.

TABLE-US-00002 TABLE 2 Reagents Amount Buffer III 25 l T4 DNA ligase 1 l

[0038] The pre-mixture was mixed thoroughly and centrifuged for 5 s at room temperature instantly. Then, the pre-mixture was added to the incubated reaction mixture, and 2 l of sequencing linkers was added on the wall of the same reaction tube, followed by centrifugation for 5 s at room temperature instantly. After rapid mixing, the mixture was centrifuged for 5 s at room temperature instantly, and bubbles were removed by slight flicks. The mixture was centrifuged for 5 s at room temperature instantly again. Then, the reaction mixture was placed in a normal PCR machine (or a thermo metal bath) to carry out the following incubation steps: 15 min at 20 C., followed by 10 min at 65 C., and finally 5 min at 4 C. The sequencing library was obtained after the incubation.

[0039] 3. purifying the library and sequencing: the obtained sequencing library was purified using a purification kit from Hangzhou Berry Genomics Diagnostic Technology Ltd. (Lot No.: R0022), and the total amount is not less than 2 fmol. Then, the purified sequencing library was loaded and sequenced on a NextSeq CN500 sequencing machine (National Medical Instrument Registration No.: 20153400460) according to the instructions of the manufactures.

[0040] 4. analyzing the sequencing results: the sequencing results were aligned with the human genome reference sequences. The detection results of chromosome copy number in samples to be tested and in normal samples are presented in FIGS. 2 and 3.

[0041] In FIGS. 2 and 3, ration indicated in the Y-axis log.sub.2(ratio) refers to the ratio between the copy number of the detected region and 2 (i.e., diploid), and the X-axis represents the whole chromosome region. The whole chromosome region was evenly divided into lots of bins, wherein the log.sub.2(ratio) value of each bin corresponds to a single dot, and the distribution of all dots was used to obtain a trend line (the grey line). The black line in FIGS. 2 and 3 represents location of centromere in the chromosome, and separates the chromosome into a short arm and a long arm. As there are no reference sequences for the short arms of chromosomes 13 and 22 in the current human genome reference sequences that can be used for data analysis, the sequencing results related to the short arms of chromosomes 13 and 22 cannot be analyzed, resulting in the absence of corresponding log.sub.2(ratio) values in these regions.

[0042] Specifically, FIG. 2 shows the detection results of copy number in the chromosome 13 region of a DNA sample to be tested (A) and a normal sample (B). It is clear that for the DNA sample to be tested, the log.sub.2(ratio) value of the long arm of chromosome 13 is around 0.585 (=log.sub.21.5), indicating that at least there are three copies for long arm part of the chromosome 13. That is, the DNA sample to be tested has Patau Syndrome (T13). Meanwhile, for the normal sample, the log.sub.2(ratio) value of the long arm of chromosome 13 is 0(=log.sub.21), indicating that the chromosome 13 has only two copies. The DNA sample to be tested was detected by SNP-array, which confirmed the presence of three copies of chromosome 13 long arm. In other words, the detection results obtained using the library construction method and kit of the present invention are consistent with the clinical detection results.

[0043] The inventors also used the method and kit of the present invention to construct second libraries for a second DNA sample to be tested and a normal sample and then performed sequencing as indicated above. The results are shown in FIG. 3. It is clear that for this DNA sample to be tested, the log.sub.2(ratio) value of chromosome 22 in the 20M region is around 1(=log.sub.20.5), indicating that this region has only one copy. When aligned to the human genome reference sequences, this region with only one copy corresponds to the 22q11.21-q11.23 region. Thus, the DNA sample to be tested was confirmed to have a chromosome 22q11.2 microdeletion syndrome. Meanwhile, for the normal sample, the log.sub.2(ratio) value of the long arm of chromosome 22 is 0(=log.sub.21), indicating that there is no deletion in 22q11.2 region of chromosome 22. The DNA sample to be tested was detected by SNP-array, which confirmed the presence of microdeletion in 22q11.2 region of chromosome 22. In other words, the detection results obtained using the library construction method and kit of the present invention are consistent with the clinical detection results.

[0044] This example shows that the library construction method and kit of the present invention can be used for constructing a sequencing library which allows the accurate detection of chromosome copy number variation by high throughput sequencing.

Example 2: The Effects of Initial Amount of DNA on the Concentration of Sequencing Library

[0045] Samples with various initial amounts of DNA were used to prepare sequencing libraries according to the library construction and purification method of Example 1, and the total amount of obtained libraries were measured. The results are represented in Table 3.

TABLE-US-00003 TABLE 3 The DNA initial amounts and the total amounts of finally obtained libraries DNA initial Total amount of DNA initial Total amount of amount (ng) library (fmol) amount (ng) library (fmol) <5 0.142 150 2.752 <5 0.2 200 2.348 <5 0.21 200 1.782 <5 0.108 220 2.462 <5 0.129 220 1.758 <5 0.169 240 2.078 5 1.053 240 1.935 5 1.592 260 2.149 10 2.776 260 2.228 10 2.401 280 1.405 50 2.97 280 1.585 50 2.988 290 1.982 100 2.318 290 1.812 100 3.11 300 1.04 150 2.477 300 0.568

[0046] Table 3 shows that when the DNA initial amount is between 10 and 260 ng, the total amounts of the obtained libraries generally are greater than 2 fmol, which satisfies the requirements for sequencing. However, when the DNA initial amount is too low or too high, the total amounts of the obtained libraries are less than 2 fmol, which is not optimal for sequencing. Thus, in the present invention, the DNA initial amount used for preparing a sequencing library preferably is 10-260n1.812 g.

[0047] It should be note that the above examples are merely the preferred embodiments of the invention and are not intended to limit the invention. Those skilled in the art know there can be various modifications and changes. Those skilled in the art will understand that any modification, equivalent replacement and improvement within the spirit and principle of the invention will be encompassed in the protection scope of the invention.

REFERENCES

[0048] Nagaoka S I, Hassold T J, and Hunt P A. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat. Rev. Genet. 2012, 13: 493e504 [0049] Nord A, Salipante S J, Pritchard C. Chapter 11Copy Number Variant Detection Using Next-Generation Sequencing. Clinical Genomics, 2015, 8:165-187 [0050] Yunis J. High-resolution chromosome analysis in clinical medicine. Prog. Clin. Pathol., 1978, 7: 267-288 [0051] Trask B J. Human cytogenetics: 46 chromosomes, 46 years and counting. Nat. Rev. Genet. 2002, 3: 769e778 [0052] Trask B J. Fluoresence in situ hybridization: application in cytogenetics and gene mapping. Trends Gnent. 1991, 7:149-154. [0053] Manning M, and Hudgins L. Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genet. Med. 2010, 12(11):742-745. [0054] Breman A, Pursley A N, Hixson P, Bi W, Ward P, Bacino C A, Shaw C, Lupski J R, Beaudet A, Patel A, Cheung S W, Van den Veyver I: Prenatal chromosomal microarray analysis in a diagnostic laboratory: experience with >1000 cases and review of the literature. Prenat. Diagn. 2012, 32: 351e361. [0055] Xuan J, Yu Y, Qing T, Guo L, Shi L. Next-generation sequencing in the clinic: promises and challenges. Cancer Lett. 2013, 340(2):284-295. [0056] Yoon S, Xuan Z, Makarov V, Ye K, Sebat J. Sensitive and accurate detection of copy number variants using read depth of coverage. Genome Res. 2009, 19(9):1586-1592. [0057] Mason-Suares H, Landry L, Lebo M S. Detecting Copy Number Variation via Next Generation Technology. Curr. Genet. Med. Report. 2016, 4 (3): 1-12. [0058] van Dijk E L, Jaszczyszyn Y, Thermes C. Library preparation methods for next-generation sequencing: tone down the bias. Exp. Cell Res. 2014, 322(1):12-20. [0059] Head S R, Komori H K, LaMere S A, Whisenant T, Van Nieuwerburgh F, Salomon D R, Ordoukhanian P1. Library construction for next-generation sequencing: overviews and challenges. Biotechniques. 2014, 56(2):61-64. [0060] Goodwin S, McPherson J D, McCombie W R. Coming of age: ten years of next-generation sequencing technologies. Nat. Rev. Genet. 2016, 17(6):333-351.