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USing cell-free DNA fragment size to determine copy number variations

A copy number, fragment technique used in the field of determining copy number variation using cell-free DNA fragment size

Active Publication Date: 2018-03-02
VERINATA HEALTH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Limitations of existing non-invasive prenatal diagnostic methods, which include insufficient sensitivity stemming from limited levels of cfDNA, and sequencing bias stemming from the inherent nature of genomic information, are the continuing need to provide any or all specificity , sensitivity, and applicability to reliably diagnose copy number alterations in a variety of clinical settings as the basis for a non-invasive approach

Method used

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  • USing cell-free DNA fragment size to determine copy number variations
  • USing cell-free DNA fragment size to determine copy number variations
  • USing cell-free DNA fragment size to determine copy number variations

Examples

Experimental program
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Embodiment 1

[0566] Example 1: Preparation and sequencing of initial and enriched sequencing libraries

[0567] a. Preparation of sequencing library - abbreviated protocol (ABB)

[0568] All sequencing libraries, naive and enriched, were prepared from approximately 2 ng of purified cfDNA extracted from maternal plasma. Use NEBNext TM The reagents of DNA Sample Prep DNA Reagent Set 1 (Part No.E6000L; NewEngland Biolabs, Ipswich, MA) were used for library preparation as follows Because cell-free plasma DNA is fragmented in nature, no further fragmentation by nebulization or sonication was performed on plasma DNA samples. according to End Repair Module by mixing cfDNA with NEBNext in a 1.5ml centrifuge tube TM DNA Sample Prep DNA Reagent Set 1 provided in 5 μl 10X phosphorylation buffer, 2 μl deoxynucleotide solution mix (10 mM each dNTP), 1 μl 1:5 diluted DNA polymerase I, 1 μl T4 DNA polymerase and 1 μl T4 polynuclear Nucleotide kinase incubation for 15 min at 20°C converted ov...

Embodiment 2

[0578] Non-invasive prenatal testing using fragment size

[0579] introduce

[0580] Since its commercial launch in late 2011 and early 2012, noninvasive prenatal testing (NIPT) of cell-free DNA (cfDNA) in maternal plasma has rapidly become the method of choice for screening pregnant women at high risk for fetal aneuploidy. This method is mainly based on the isolation and sequencing of cfDNA from maternal plasma and counting the number of cfDNA fragments aligned to specific regions of the reference human genome (ref: Fan et al, Lo et al). These DNA sequencing and molecular counting methods allow high-precision determination of the relative copy number of each chromosome in the genome. High sensitivity and specificity for the detection of trisomies 21, 18 and 13 have been reproduced in several clinical studies (ref, citing Gil / Nicolaides meta-analysis).

[0581] More recently, additional clinical studies have shown that this approach can be extended to the general obstetr...

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Abstract

Disclosed are methods for determining copy number variation (CNV) known or suspected to be associated with a variety of medical conditions. In some embodiments, methods are provided for determining copy number variation (CNV) of fetuses using maternal samples comprising maternal and fetal cell free DNA. In some embodiments, methods are provided for determining CNVs known or suspected to be associated with a variety of medical conditions. Some embodiments disclosed herein provide methods to improve the sensitivity and / or specificity of sequence data analysis by deriving a fragment size parameter, such as a size-weighted coverage or a fraction of fragments in a size range. In some embodiments, the fragment size parameter is adjusted to remove within-sample GC-content bias. In some embodiments, removal of within-sample GC-content bias is based on sequence data corrected for systematic variation common across unaffected training samples. Also disclosed are systems and computer program products for evaluation of CNV of sequences of interest.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 091,380, filed December 12, 2014, entitled USING CELL-FREE DNA FRAGMENT SIZETO DETERMINE COPY NUMBER VARIATIONS, for all purposes Its entirety is incorporated herein by reference. Background of the invention [0003] One of the key tasks of human medical research is the discovery of genetic abnormalities that have adverse health consequences. In many cases, specific genes and / or key diagnostic markers were identified in portions of the genome present at abnormal copy numbers. For example, extra or missing copies of entire chromosomes are frequently occurring genetic lesions in prenatal diagnosis. In cancer, loss or proliferation of copies of entire chromosomes or segments of chromosomes and higher levels of amplification of specific regions of the genome are common events. [0004] Most information on copy number ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6809C12Q1/6869G06F19/18G06F19/22G16B20/10G16B20/20G16B30/10
CPCG16B20/00G16B30/00C12Q1/6809C12Q1/6869G16B20/20G16B20/10G16B30/10C12Q2535/122C12Q2537/16C12Q2537/165C12Q2545/101
Inventor D.I.丘杜瓦C.巴巴西奥鲁S.杜恩瓦尔德D.A.康斯托克R.P.拉瓦
Owner VERINATA HEALTH INC
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