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Simultaneous determination of aneuploidy and fetal fraction

a technology of fetal fraction and simultaneous determination, applied in the field of diagnostics, can solve the problems of false negative results and a great risk to the pregnancy

Inactive Publication Date: 2011-09-15
VERINATA HEALTH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for simultaneously determining the presence or absence of fetal aneuploidy and the relative amount of fetal nucleic acid in a maternal sample. The method involves enriching the sample for polymorphic target nucleic acids and sequencing the enriched material using a variety of techniques. The simultaneous detection of both fetal aneuploidy and fetal fraction can be useful in prenatal diagnostics. The method is noninvasive and can be performed using a single sequencing library.

Problems solved by technology

Imaging techniques such as ultrasonography, magnetic resonance imaging and fetal echocardiography are useful for the identification of structural abnormalities of the fetus Amniocentesis, chronic villus sampling and fetal blood sampling provide fetal cells and tissues for the analysis of chromosomal, genetic and biochemical abnormalities, but are invasive and pose great risk to the pregnancy.
Given the relatively low concentration of fetal circulating nucleic acids, false negative results can arise if there is insufficient starting nucleic acid for analysis.

Method used

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  • Simultaneous determination of aneuploidy and fetal fraction
  • Simultaneous determination of aneuploidy and fetal fraction
  • Simultaneous determination of aneuploidy and fetal fraction

Examples

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example 1

Sample Processing and cfDNA Extraction

[0163]Peripheral blood samples were collected from pregnant women in their first or second trimester of pregnancy and who were deemed at risk for fetal aneuploidy. Informed consent was obtained from each participant prior to the blood draw. Blood was collected before amniocentesis or chorionic villus sampling. Karyotype analysis was performed using the chorionic villus or amniocentesis samples to confirm fetal karyotype.

[0164]Peripheral blood drawn from each subject was collected in ACD tubes. One tube of blood sample (approximately 6-9 mL / tube) was transferred into one 15-mL low speed centrifuge tube. Blood was centrifuged at 2640 rpm, 4° C. for 10 min using Beckman Allegra 6 R centrifuge and rotor model GA 3.8.

[0165]For cell-free plasma extraction, the upper plasma layer was transferred to a 15-ml high speed centrifuge tube and centrifuged at 16000×g, 4° C. for 10 min using Beckman Coulter Avanti J-E centrifuge, and JA-14 rotor. The two centri...

example 2

Preparation and Sequencing of Primary and Enriched Sequencing Libraries

[0167]a. Preparation of Sequencing Libraries

[0168]All sequencing libraries i.e. primary and enriched libraries, were prepared from approximately 2 ng of purified cfDNA that was extracted from maternal plasma. Library preparation was performed using reagents of the NEBNext™ DNA Sample Prep DNA Reagent Set 1 (Part No. E6000L; New England Biolabs, Ipswich, Mass.), for Illumina® as follows. Because cell-free plasma DNA is fragmented in nature, no further fragmentation by nebulization or sonication was done on the plasma DNA samples. The overhangs of approximately 2 ng purified cfDNA fragments contained in 40 μl were converted into phosphorylated blunt ends according to the NEBNext® End Repair Module by incubating in a 1.5 ml microfuge tube the cfDNA with 5 μl 10× phosphorylation buffer, 2 μl deoxynucleotide solution mix (10 mM each dNTP), 1 μl of a 1:5 dilution of DNA Polymerase I, 1 μl T4 DNA Polymerase and 1 μl T4 ...

example 3

Analysis of Sequencing Data for the Determination of Aneuploidy and Fetal Fraction

[0170]a. Analysis of Sequencing Data for the Determination of Aneuploidy

[0171]Upon completion of sequencing of the sample, the Illumina “Sequencer Control Software” transferred image and base call files to a Unix server running the Illumina “Genome Analyzer Pipeline” software version 1.51. The Illumina “Gerald” program was run to align sequences i.e. 36 bp reads, to the hg18 reference human genome provided by National Center for Biotechnology Information (NCBI36 / hg18, available on the world wide web at genome.ucsc.edu / cgi-bin / hgGateway?org=Human&db=hg18&hgsid=166260105). The sequence data generated from the above procedure that uniquely aligned to the genome was read from Gerald output (export.txt files) by a program (c2c.p1) running on a computer running the Linux operating system. Sequence alignments with base mis-matches were allowed and included in alignment counts only if they aligned uniquely to ...

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Abstract

The invention provides compositions and methods for simultaneously determining the presence or absence of fetal aneuploidy and the relative amount of fetal nucleic acids in a sample obtained form a pregnant female. The method encompasses the use of sequencing technologies and exploits the occurrence of polymorphisms to provide a streamlined noninvasive process applicable to the practice of prenatal diagnostics.

Description

CROSS REFERENCE[0001]This application claims priority to U.S. Provisional Application Ser. No. 61 / 296,358 entitled “Methods for Determining Fraction of Fetal Nucleic Acids in Maternal Samples”, filed on Jan. 19, 2010; U.S. Provisional Application Ser. No. 61 / 360,837 entitled “Methods for Determining Fraction of Fetal Nucleic Acids in Maternal Samples”, filed on Jul. 1, 2010; U.S. Provisional Application Ser. No. 61 / 407,017 entitled “Method for Determining Copy Number Variations”, filed on Oct. 26, 2010; and U.S. Provisional Application Ser. No. 61 / 455,849 entitled “Simultaneous determination of Aneuploidy and Fetal Fraction”, filed on Oct. 26, 2010; which are incorporated herein by reference in their entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 22, 2011, is named 32477820.txt and is 238,522 bytes in size....

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B30/00C40B40/06
CPCC12Q1/6806C12Q1/6869C12Q1/6883C12Q2600/106C12Q1/6809C12Q2537/16C12Q2537/165C12Q2545/101C40B30/00C12Q1/68C12Q1/6827G16B99/00G16B30/00C12Q1/6872G16B30/10G16B20/10C12Q2600/112G16H10/40
Inventor QUAKE, STEPHENRAVA, RICHARD P.CHINNAPPA, MANJULACOMSTOCK, DAVID A.HEILEK, GABRIELLE
Owner VERINATA HEALTH INC
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