Reagent kit and library construction method for detection of free dna in peripheral blood of pregnant women
A technology for maternal peripheral blood and DNA library, applied in the field of molecular biology, can solve the problems of high-throughput gene detection economy, large amount of sequencing reagents, and high sequencing cost, so as to shorten sequencing time, improve sequencing efficiency, and save reagents Effect
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Embodiment 1
[0064] Example 1 Using the method of the present invention to construct a DNA library for next-generation sequencing
[0065] (1) Preparation of DNA fragments to be sequenced
[0066] Using the DNA extraction kit from TIANGEN company to extract free DNA in the plasma of pregnant women, the steps are as follows:
[0067] a. Take 600μL of plasma into a 2.0mLEP tube. Add 10 μL proteinase K, vortex to mix;
[0068] b. Add 600 μL buffer GB and 6 μL Carrier RNA, vortex to mix, and centrifuge at 12000 rpm for 30 seconds. 56°C water bath for 10 minutes. Stand at room temperature for 5 minutes, centrifuge at 2000rpm for 10 seconds;
[0069] c. Add 200 μL of frozen absolute ethanol and invert up and down 6 times. Let stand at room temperature for 5 minutes, and centrifuge at 2000 rpm for 10 seconds.
[0070] d. Transfer the solution in step d to a 750 μL adsorption column, centrifuge at 10000rp for 30 seconds, discard the waste liquid, put the adsorption column back into the colle...
Embodiment 2
[0113] Example 2 Detection of the quality of the DNA library for next-generation sequencing
[0114] The DNA library constructed in Example 1 was used to detect the fragment size of the library using an Agilent 2100 bioanalyzer.
[0115] Use the Agilent 2100 Bioanalyzer to test the library, and you can know whether the constructed library fragments meet the above library quality standards. For the test results, see figure 2 ,From figure 2 can be understood in
[0116] ① There are mainly two fragments of different sizes: 480bp±10% and 660bp±10%;
[0117] ② The proportion of 480bp±10% fragments is about 50%-70%, and the proportion of 660bp±10% fragments is about 30%-50%.
[0118] It shows that the construction of the DNA library obtained in Example 1 is qualified.
Embodiment 3
[0119] Example 3 Using Illumina NextSeq500 to Sequence the DNA Library for Next Generation Sequencing
[0120] The DNA library for next-generation sequencing constructed in Example 1 was subjected to single-end sequencing and paired-end sequencing respectively, and the reagent used for sequencing was NextSeq TM 500High Output v2Kit, the sequencing results are as follows Figure 3-6 shown.
[0121] From image 3 and Figure 4 From the base distribution map, we can know that the results of single-end sequencing and paired-end sequencing are basically consistent with the above-mentioned DNA library for next-generation sequencing.
[0122] From Figure 5 and Figure 6 From the quality distribution diagram, we can know that the quality curve is smooth and there is no falling point.
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