Method for non-invasive antenatal determination of aneuploidy by targeting next generation sequencing with biallelic SNP

A biallelic, aneuploidy technique for non-invasive prenatal testing

Inactive Publication Date: 2019-07-05
MEDTIMES MOLECULAR LAB LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, studying STRs on the Y chromosome increases accuracy and sensitivity, but these methods are only applicable to male fetuses and inevitably require sex determination before NIPT

Method used

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  • Method for non-invasive antenatal determination of aneuploidy by targeting next generation sequencing with biallelic SNP
  • Method for non-invasive antenatal determination of aneuploidy by targeting next generation sequencing with biallelic SNP
  • Method for non-invasive antenatal determination of aneuploidy by targeting next generation sequencing with biallelic SNP

Examples

Experimental program
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preparation example Construction

[0120] In one embodiment, an amplicon-based library preparation method is used, ie, target-specific primers are used to selectively amplify and thus enrich for target sequences. In one embodiment, the amplicon-based method comprises the steps of:

[0121] 1. Free DNA extracted from end repair and A-tail processing;

[0122] 2. Ligation of cell-free DNA with platform-specific adapter sequences, index sequences, and unique molecular identifiers (UMI);

[0123] 3. Wash away unconnected debris;

[0124] 4. Amplification of ligated free DNA using target-specific primers;

[0125] 5. Amplify the enriched fragments using universal primers; and

[0126] 6. Purification of the amplified target sequence.

[0127] In one embodiment, the index sequence is added to the target sequence in a post-enrichment amplification and ligation step.

[0128] As used herein, libraries are prepared based on amplicons, which are short synthetic DNA fragments that allow DNA ligated to the adapters to...

example 1

[0331] Sample Preparation - Purification of Cell-Free DNA from Maternal Blood or Plasma Samples

[0332] This example illustrates the use of a The Rapid Sample Concentrator (RSC) extracts cell-free DNA from maternal whole blood or plasma samples and uses Example of Fluorimeter Measurement of Extracted Genomic DNA Concentration.

[0333] 10 mL whole blood samples were collected from pregnant subjects and stored in cfDNA blood tubes. Samples were then processed according to the following scheme:

[0334] Prepare plasma

[0335] 1. Centrifuge whole blood from cfDNA blood tubes at 3000 rpm for 5 minutes.

[0336] 2. Aliquot all plasma and centrifuge collected plasma at 14000 rpm for 10 minutes.

[0337] 3. Collect the supernatant in a new 2 mL screw cap tube and store at 4°C until further use, or -20°C for long-term storage.

[0338] Binding of circulating nucleic acids to magnetic resins

[0339] 1. Add 2mL of Binding Buffer to a 50mL centrifuge tube.

[0340] 2. Ad...

example 2

[0384] Hybridization-Based Targeted Enrichment-Library Preparation Protocol

[0385] In the present invention, hybridization-based library preparation was built on the SeqCap manufacturer's protocol. In the following protocols and procedures, minor differences from the manufacturer's version were introduced to customize and optimize the workflow for better sequence reads at subsequent stages.

[0386] The process includes DNA fragmentation, end repair and A-tail processing, adapter ligation, library amplification (i.e., pre-enrichment amplification), post-amplification cleanup, and sample hybridization with the SeqCap probe pool (i.e., target enrichment amplification). ) and post-hybridization amplification (i.e. post-enrichment amplification). The detailed plan is as follows:

[0387] A. Bring AMPure XP beads to room temperature

[0388] B. Prepare DNA (free DNA is recommended to use 1ng-1μg)

[0389] 1. Maximize volume - 50 µL cfDNA input

[0390] C. End repair and A-...

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Abstract

The present invention provides a method for non-invasive pre-natal test (NIPT) for determining the probability of aneuploidy in a fetus. The method includes a step of carrying out absolute or relativequantification platform quantification and analysis of the autosomal single nucleotide polymorphism (SNP) to determine the probability of fetal aneuploidy. In one embodiment, the method comprises thefollowing steps: obtaining a blood sample containing free DNA (cfDNA) from a pregnant woman; using the extracted DNA to prepare a nucleic acid library containing a plurality of target biallele autosomal single nucleotide polymorphisms (SNP) (i.e., target SNPs) by using a target enrichment method; carrying out targeted next generation sequencing (NGS) by using the prepared library, obtaining allele counting of a target SNP in the free DNA, and determining the probability of fetal aneuploidy.

Description

technical field [0001] The present invention relates to the field of non-invasive prenatal testing (NIPT) for determining the probability of aneuploidy. In particular, the present invention relates to the non-invasive prenatal determination of trisomy 13 (T13), trisomy 18 (T18) and trisomy 21 (T21). Background technique [0002] Pregnant women are usually advised to test for fetal chromosomal abnormalities at 6-12 weeks of gestation. The goal of this test is to determine the probability that the fetus will develop aneuploidy (abnormal number of chromosomes). Trisomy 21 (T21) has been shown to cause Down syndrome, trisomy 18 (T18) may cause Edwards syndrome, and trisomy 13 (T13) may cause Patau syndrome. When a fetus is found to be at risk for chromosomal abnormalities, early prenatal testing of the fetus can provide parents with a choice to continue the pregnancy or not. It is also a way to check whether a baby is at risk for chromosomal abnormalities. [0003] Unlike pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2535/122C12Q2537/165C12Q1/6869G16B20/10G16B20/20G16B25/00G16B40/00G16B30/00
Inventor 谭楚颖陈伊文周俊健
Owner MEDTIMES MOLECULAR LAB LTD
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