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Method of determining cfDNA ratio of donor source in receptor cfDNA sample

A sample-in-receptor technology, applied in the field of biological information and biological detection, can solve the problems of difficult cfdDNA content, inability to be widely used in clinical practice, lack of genetic information, etc.

Active Publication Date: 2017-03-29
BGI GUANGZHOU MEDICAL LAB CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At the beginning, research in this area focused on gender-mismatched organ transplantation, that is, female recipients received organs from male donors, and the content of donor-derived cfDNA in plasma was determined by detecting Y-chromosome-specific genes, which was subject to many limitations. Not widely used clinically
[0006] Furthermore, there is often a lack of pre-transplantation samples, especially donor samples, and the lack of genetic information of the donor also makes it difficult to determine the content of cfdDNA
[0007] Existing methods for determining the amount of donor cell-free DNA (cfdDNA) in recipients, and for detecting organ transplant rejection, need to be improved or supplemented

Method used

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  • Method of determining cfDNA ratio of donor source in receptor cfDNA sample
  • Method of determining cfDNA ratio of donor source in receptor cfDNA sample
  • Method of determining cfDNA ratio of donor source in receptor cfDNA sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Experimental methods for obtaining the first sequencing data generally include:

[0066] (1) Target SNP site design

[0067] Since this example method requires a high sequencing depth of SNP sites, which is 200× or more on average, the use of ordinary chips in this method will cause a lot of data waste and greatly increase the detection cost. With the idea that the frequency (MAF) value is closer to 0.5, a small SNP chip was independently designed and synthesized for target area capture.

[0068] The main ways to obtain the target SNP site are as follows:

[0069] 1. The ALFRED allele frequency database is filtered according to the interval of heterozygosity from 0.48 to 0.5 to obtain 946 SNP sites;

[0070] 2. In the 1000Genomes database, filter according to the frequency of 0.5 in the EAS supergroup sub-library, and then filter according to the average frequency of all groups in the library from 0.4 to 0.5, and obtain a total of 2263 SNP sites;

[0071] 3. HapMa...

Embodiment 2

[0128] The design idea of ​​the embodiment is as follows: take 2 normal human blood samples (taken from volunteers), one is the donor and the other is the recipient, mix the samples to be tested, and carry out the simulation experiment. Separation of blood cells and plasma from the blood sample taken, the recipient blood cells (donor blood cells are not required) to extract genomic DNA, interrupt the DNA and perform target region capture sequencing for genotyping; after donor and recipient plasma extract cfDNA, Agelint 2100 The concentration was measured, and the cfDNA of the donor and recipient were artificially mixed according to the ratio of 3.5%, 5.5%, 8%, and 10%, and then the mixed cfDNA was constructed for library capture and sequencing (the sequencer used in this example was the BGISEQ-100 sequencing platform), using To test the reliability of this experimental method. According to the experimental steps in Example 1, the steps in this example are also divided into two...

Embodiment 3

[0323] In order to verify the technical feasibility of using the allele frequency detection method, a simulation verification experiment with known donor ratio was carried out. Taking the BGISEQ-100 sequencing platform as an example, in this embodiment, a normal recipient (sample name R) blood cell sample is selected for target region capture sequencing, and the recipient plasma mixed with donor plasma DNA is also subjected to target region capture sequencing. The proportions were 3.5%, 5.5%, 8%, and 10%, respectively, and the sample names were named according to the mixing ratio. The effective data of the sequencing were compared through tmap comparison, BamDuplicates deduplication, quality control (QC), recipient blood cell genotyping, and recipient blood cell genotyping. The blood plasma frequency statistics and the donor ratio calculation were carried out, and the test reports of the donor content at the 4 blood collection points were finally obtained to evaluate the degree...

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Abstract

The invention discloses a method of determining a cfDNA ratio of a donor source in a receptor cfDNA sample. The method comprises acquiring first and second sequencing data comprising at least one part of sequencing results of the receptor gDNA and the receptor cfDNA, respectively comparing the first and second sequencing data and a reference sequence to obtain first and second comparison results, carrying out SNP detection based on the first comparison result to obtain a first classification result comprising a number of primary homozygous SNPs expressed as AA, and determining a proportion of cfDNA of the donor source based on the amount of a second reading section matching with secondary homozygous SNP in the second comparison result, wherein the secondary homozygous SNP is least one part of the allele A-nonsupport primary homozygous SNP of the second reading section matching with the site. The method can accurately determine the content of the donor source cfDNA in a receptor cfDNA without a donor genetic sample.

Description

technical field [0001] The present invention relates to the fields of biological information and biological detection. Specifically, the present invention relates to a method for determining the proportion of donor-derived cfDNA in a recipient cfDNA sample, a device for determining the proportion of donor-derived cfDNA in a recipient cfDNA sample, A method for monitoring immune rejection and a device for monitoring immune rejection. Background technique [0002] Organ and tissue transplantation is one of the most important medical achievements in the 20th century. At present, transplantation has become the most effective treatment for the terminal stage of tissue and organ failure. Monitoring the immune rejection of organ transplant patients is an important means to improve the long-term survival rate of organ transplant patients. At present, the diagnosis of acute rejection mainly relies on biopsy of transplanted organs, for example, myocardial and endocardial biopsy after...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/34
CPCC12Q1/6869C12Q2535/125C12M1/00C12M1/34C12Q1/68G01N33/00G01N33/48G01N33/49G16Z99/00
Inventor 曾柳红袁盛建杨青张纪斌叶明芝
Owner BGI GUANGZHOU MEDICAL LAB CO LTD
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