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Method for determining the presence or absence of different aneuploidies in a sample

An aneuploidy, sample technique, used in diagnostics

Active Publication Date: 2013-03-27
VERINATA HEALTH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, multiple limitations of existing methods, which include insufficient sensitivity from limited levels of cfDNA, and sequencing bias from the inherent nature of genomic information dictate a continuing need for noninvasive methods that will Provide any or all of specificity, sensitivity, and applicability to reliably diagnose copy number alterations in a variety of clinical settings

Method used

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  • Method for determining the presence or absence of different aneuploidies in a sample
  • Method for determining the presence or absence of different aneuploidies in a sample
  • Method for determining the presence or absence of different aneuploidies in a sample

Examples

Experimental program
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example 1

[0264] Sample Processing and DNA Extraction

[0265] Peripheral blood samples were collected from multiple pregnant women considered at risk for fetal aneuploidy during their first and second trimesters. Consent was obtained from each participant prior to blood draw. Blood was collected prior to amniocentesis or chorionic villus sampling. Karyotyping was performed using chorionic villus or amniocentesis samples to confirm fetal karyotyping.

[0266] Peripheral blood drawn from each subject was collected in ACD tubes. Transfer one tube of blood sample (approximately 6-9 mL / tube) to a 15-mL low-speed centrifuge tube. Blood was centrifuged at 2640 rpm, 4°C for 10 minutes using a Beckman Allegra 6R centrifuge and rotor model GA 3.8.

[0267]For cell-free plasma extraction, the supernatant plasma was transferred to a 15-mL high-speed centrifuge tube and centrifuged at 16000 x g, 4°C for 10 minutes using a Beckman Coulter Avanti J-E centrifuge with a JA-14 rotor. These two cent...

example 2

[0274] Dosage and variation for chromosomes 13, 18, 21, X, and Y

[0275] To examine the extent of interchromosomal variability and intersequence determination variability in the number of mapped sequence tags for all chromosomes, plasma cfDNA obtained from the peripheral blood of 48 volunteer pregnant subjects was extracted and as in example The instructions in 1 were sequenced and analyzed as follows.

[0276] The total number of sequence tags mapped to each chromosome (sequence tag density) was determined. Alternatively, the number of mapped sequence tags can be normalized to the length of the chromosome to yield a sequence tag density ratio. Normalization to chromosome length is not a required step, but can be done separately to reduce the number of digits in a number, thereby simplifying it for human interpretation. Chromosome lengths that can be used to normalize these sequence tag counts can be those provided at the world wide web site genome.ucsc.edu / goldenPath / stats...

example 3

[0295] Fetal aneuploidy diagnosed using normalizing chromosomes

[0296] To implement the use of chromosome dosage to assess aneuploidy in biological test samples, maternal blood test samples were obtained from pregnant volunteers and cfDNA was prepared, sequenced and analyzed as described in Examples 1 and 2.

[0297] Trisomy 21

[0298] Table 4 provides the calculated doses for chromosome 21 in an exemplary test sample (#11403). The calculated threshold for a positive diagnosis of T21 was set at >2 standard deviations from the mean of these qualified (normal) samples. The diagnosis of T21 is given based on the chromosome dose in the test sample being greater than a set threshold. Chromosomes 14 and 15 were used as normalizing chromosomes in separate calculations to show that either the chromosome with the lowest variability (e.g., chromosome 14) or the chromosome with the greatest discriminability (e.g., chromosome 15) could be used to identify aneuploidy. Thirteen T21 s...

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Abstract

The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the present method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and / or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample. Any aneuploidy can be determined from sequencing information that is obtained by sequencing only once the nucleic acids of a test sample.

Description

field of invention [0001] The present invention relates generally to the field of diagnostics and provides methods for determining variation in the amount of nucleic acid sequences in a mixture of nucleic acids derived from different genomes. In particular, the method is suitable for the implementation of non-invasive prenatal diagnosis and for the diagnosis and monitoring of metastatic progression in cancer patients. Background of the invention [0002] One of the key endeavors in human medical research is the discovery of genetic abnormalities that are extremely important for adverse health outcomes. In many cases, specific genes and / or key diagnostic markers have been identified in multiple parts of the genome in abnormal copy numbers. For example, in prenatal diagnosis, extra or missing copies of entire chromosomes are a frequently occurring genetic lesion. In cancer, copy loss or multiplication of entire chromosomes or segments of chromosomes, and higher levels of amp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q1/6883C12Q1/6806C12Q2545/101C12Q2535/122C12Q2531/113C12Q1/6827C12Q1/6872G16B5/00G16B10/00G16B15/00G16B20/00G16B25/00G16B30/00G16B40/00G16B45/00G16B50/00G16B99/00
Inventor 里查德·P·拉瓦大卫·A·康斯托克布莱恩·K·利思
Owner VERINATA HEALTH INC
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