Simplified analysis method of dna and cell free dna and uses thereof
AssigneeYunnan Daoran Science and Technology Ltd.
InventorsDonald Arthur Leigh, David Stephen Cram, Li Wang
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Abstract
Provided are an amplification-free method for the preimplantation genetic testing (PGT) of in-vitro fertilization (IVF) embryos, an amplification-free method of generating a library for the PGT, and a method of identifying the genetic background of IVF embryos. Provided are also a method of determining the degree of non-embryonic DNA contamination, and an amplification-free method of generating a library of cell-free DNAs (cfDNAs) from a biological material. Similarly provided are a method for enrichment of methylated fragments from the amplification free library, and a method for identifying methylation profiles and/or changes of same, in samples of different sources.
Claims56
An amplification-free method for analyzing cell-free DNA (cfDNA) in a biological sample comprising
- i) providing a sample containing cell-free DNA (cfDNA);
- ii) repairing the cfDNA fraction to obtain single base overhang fragments;
- iii) adding a barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library for sequencing;
- and
- iv) sequencing the barcoded library or prior to sequencing, processing through further enrichment steps.
An amplification-free method for the preimplantation genetic testing (PGT) of in-vitro fertilization (IVF) embryos comprising
- i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of an IVF embryo;
- ii) repairing the cfDNA fraction to obtain blunt ended fragments;
- iii) adding a barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library for sequencing;
- and
- iv) sequencing the barcoded library.
An amplification-free method of generating a library for the preimplantation genetic testing (PGT) of in-vitro fertilization (IVF) embryos comprising
- i) providing a sample containing cell-free DNA (cfDNA) from the spent medium in the culture of an IVF embryo;
- ii) repairing the cfDNA to obtain blunt ended fragments;
- and
- iii) adding a barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library for sequencing.
A method of identifying the genetic background of in-vitro fertilization (IVF) embryos comprising
- i) providing a sample containing cell-free DNA (cfDNA) from the spent medium in the culture of an IVF embryo;
- ii) repairing the cfDNA to obtain blunt ended fragments;
- and
- iii) adding a barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library;
- and
- iv) determining the genome-wide heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library.
An amplification-free method of generating a library of cell-free DNAs (cfDNAs) from a biological material, the method comprising
- i) providing a sample containing cell-free DNA (cfDNA) from the biological material;
- ii) repairing the cfDNA to obtain blunt ended fragments;
- and
- iii) adding a barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library for sequencing.
An amplification-free method for preparing the sample for methylation profile analysis, the method comprising
- i) providing a sample containing cell-free DNA (cfDNA) from the biological material;
- ii) repairing the cfDNA to obtain single base overhang fragments;
- and
- iii) adding a directional barcoded adapter to each of the ends of the repaired cfDNA, thereby generating a barcoded library and avoiding PCR amplification which destroys the methylation patterns by converting methyl-C to C.
Dependencies are inferred from the claim text.