A method for increasing the proportion of fetal free DNA in the plasma free DNA sequencing library of pregnant women

A technology for plasma and pregnant women, applied in the field of biotechnology genetic engineering, can solve the problems of low sequencing throughput, low fetal DNA content, and high detection cost, and achieve the effects of improving detection throughput, improving sequencing sample throughput, and accurate detection.

Active Publication Date: 2018-08-28
SUZHOU BASECARE MEDICAL DEVICE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the samples of non-invasive prenatal testing are all plasma cell-free DNA of pregnant women, but there are a large amount of maternal cell-free DNA in the cell-free DNA, and the content of fetal cell-free DNA varies greatly due to individual differences, and the content of fetal cell-free DNA in some samples is too low, resulting in accurate Reduced reliability, low sequencing throughput, and high detection costs
The ACOG guidelines emphasize that due to individual reasons, the fetal free DNA content may be too low, and the test results cannot be obtained. At this time, the possibility of repeated testing to obtain accurate results is only 50–60%. Therefore, when the fetal free DNA content is too low , the accuracy of non-invasive prenatal testing results is also relatively reduced
In order to ensure the accuracy of each sample detection, the current common practice is to increase the amount of sample detection data, thereby reducing the sample size of each sequencing run, resulting in relatively high detection costs.

Method used

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  • A method for increasing the proportion of fetal free DNA in the plasma free DNA sequencing library of pregnant women
  • A method for increasing the proportion of fetal free DNA in the plasma free DNA sequencing library of pregnant women
  • A method for increasing the proportion of fetal free DNA in the plasma free DNA sequencing library of pregnant women

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Comparison of mixed sequencing data volumes of different quantities of original libraries

[0056] experimental method

[0057] 1. Collect plasma samples from 54 pregnant women and perform cell-free DNA extraction

[0058] (1) Use EDTA anticoagulant blood collection tubes to extract 5ml of venous blood from pregnant women with a gestational age of 12-24 weeks, centrifuge the EDTA anticoagulant blood collection tubes at 4°C at 1600×g for 10 minutes; absorb all the upper plasma Dispense into 2ml centrifuge tubes, centrifuge the plasma obtained from the first centrifugation at 16000×g for 10 min at room temperature, carefully draw the upper plasma obtained from the second centrifugation into a new centrifuge tube for free DNA extraction ;

[0059] (2) Draw 600 μl of the plasma sample that has been centrifuged twice into a new 2ml centrifuge tube;

[0060] (3) Add 1000 μl Lysis Buffer, 60 μl Magnetic beads, 35 μl Proteinase K, 14 μl Acryl Carrier to 600 μl plasma in turn...

Embodiment 2

[0104]Using high-throughput sequencing technology and data analysis system to analyze and compare the proportion of fetal free DNA in the original library of plasma free DNA in pregnant women and the recovered library constructed according to the method of the present invention

[0105] experimental method

[0106] 1. Plasma sample preparation and DNA extraction

[0107] Collect 14 cases of peripheral blood samples from pregnant women who are pregnant with male fetuses, perform plasma separation and free DNA extraction according to the method in Example 1, use 1ul solution samples containing free DNA to carry out Qubit quantitative detection, and detect qualified plasma free DNA. The next step is library preparation.

[0108] 2. Library Preparation

[0109] Same as the library preparation in Example 1.

[0110] 3. Sequencing and bioinformatics analysis of the original library

[0111] Same as the original library in Example 1 for on-machine sequencing and bioinformatics an...

Embodiment 3

[0122] The method of the present invention is sensitive to the detection sensitivity of fetal cell-free DNA with different content proportions in the plasma of pregnant women

[0123] experimental method

[0124] 1. Plasma sample preparation and cell-free DNA extraction

[0125] Collect the plasma samples of pregnant women whose karyotype is known to be trisomy 21 and a male fetus after sequencing, and the content of fetal cell-free DNA in the plasma has been determined by high-throughput sequencing technology in the early stage, using the known fetal DNA content Plasma samples and non-pregnant plasma samples were mixed in different proportions to prepare mixed plasma with fetal cell-free DNA content accounting for 2%, 3%, 4%, 5% and 10% respectively. The preparation of mixed plasma is shown in Table 8 below:

[0126] Table 8

[0127]

[0128]

[0129] The mixed plasma in Table 8 was extracted according to the extraction method in Example 1, and 1 μl of the solution s...

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Abstract

The invention discloses a method for improving a proportion of fetal free DNA in a maternal plasma free DNA sequencing library. The method comprises the following steps: extracting maternal plasma free DNA; adding specific sequence tags at two ends of the maternal plasma free DNA so as to obtain plasma free DNA with maternal individual identification markings; subjecting the plasma free DNA with the maternal individual identification markings to PCR amplification so as to obtain an original library of maternal individual plasma free DNA; and recovering the maternal original library in a selected length range so as to obtain a recovered library used for high-throughput sequencing, or mixing the maternal original libraries of a plurality of different individual pregnant women so as to build the recovered library. The method provided by the invention can accurately detect a sample with excessively low content of maternal peripheral plasma fetal free DNA, reduces the sequencing data volume of every sample, improves the sample throughput of a sequencing reaction, reduces detection cost, and facilitates large-scale popularization.

Description

technical field [0001] The invention relates to the field of biotechnology genetic engineering. More specifically, it relates to a method for increasing the proportion of fetal cell-free DNA in a maternal plasma cell-free DNA sequencing library. Background technique [0002] Chromosomal aneuploidy refers to the increase or decrease in the number of one or several chromosomes in a cell compared to the normal 46 chromosomes in a human being. Changes in the number of chromosomes, especially aneuploidy, are closely related to some human diseases and also to Significant morbidity and mortality in infancy are closely related. These disorders increase with maternal age. According to literature reports, about 100,000 newborns with chromosomal abnormalities are born in China every year. Among live births, chromosomal abnormalities account for 0.3%. Chromosomal abnormalities are the main cause of a large number of birth defects. The abnormality mainly refers to the number or struct...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6855C12Q1/6869C40B50/06C12Q2531/113C12Q2525/191
Inventor 梁波孔令印申静静冒燕宣黎明刘慧敏
Owner SUZHOU BASECARE MEDICAL DEVICE CO LTD
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