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Methods of fetal abnormality detection

a technology of fetal abnormality and detection method, applied in combinational chemistry, biochemistry apparatus and processes, library member identification, etc., can solve the problem of high cost of generating sufficient data for fetal aneuploidy detection, and the sequence of a large number of polynucleotides can be expensiv

Inactive Publication Date: 2011-12-22
VERINATA HEALTH INC
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0004]In one aspect, a method for determining the presence or absence of fetal aneuploidy is provided comprising a) selectively enriching non-random polynucleotide sequences of genomic DNA from a cell-free DNA sample; b) sequencing said enriched polynucleotide sequences; c) enumerating sequence reads from said sequencing step; and d) determining the presence or absence of fetal aneuploidy based on said enumerating. In one embodiment, said selectively enriching comprises performing PCR. In another embodiment, said selectively enriching comprises linear amplification. In another embodiment, said selectively enriching comprises enriching at least 1, 5, 10, 50, 100, or 1000 non-random polynucleotide sequences from a first chromosome. In another embodiment, said selectively enriching comprises enriching at least 1, 10, or 100 polynucleotide sequences from one or more regions of a first chromosome, wherein each region is up to 50 kb. In another embodiment, said non-random polynucleotide sequences comprise sequences that are sequenced at a rate of greater than 5-fold than other sequences on the same chromosome. In another embodiment, said non-random polynucleotide sequences each comprise about 50-1000 bases. In another embodiment, said cell-free DNA sample is a maternal sample. In another embodiment, said maternal sample is a maternal blood sample. In another embodiment, said maternal sample comprises fetal and maternal cell-free DNA. In another embodiment, said cell-free DNA is from a plurality of different individuals.

Problems solved by technology

Sequencing a large number of polynucleotides to generate sufficient data for fetal aneuploidy detection can be expensive.

Method used

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Examples

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example 1

“Hot Spot” Amplification Strategy

[0099]FIG. 1 illustrates a strategy for selecting sequences from chromosome 21 for enrichment. In step 100, sequence run data was combined. Total chromosome 21 sequence reads were used (102). These samples can include reads from samples that contain trisomy 21. “Hot” and “cold” regions of sequence coverage were mapped on chromosome 21 (104). For example, the region examined can be within a 5.8 Mb Down syndrome critical region (DSCR). PCR primers are designed, which can anneal to intergenic DNA or intragenic DNA (106). The primers were designed to anneal specifically with chromosome 21. The regions to be amplified can be a hot spot region, or region to which a number of sequence reads map (108). The PCR fragments generated can be approximately 200 bp in length. Next, sequencing analysis is performed using BioAnalyzer analysis and / or PCR / probe analysis (110).

[0100]PCR primers were designed to generate amplicons of approximately 200 bp and 150 bp from c...

example 2

Chromosome Walk Strategy for Sequence Enrichment

[0108]FIG. 11 illustrates an overview of the chromosome walk strategy for sequence enrichment. A 5.8 Mbp Down syndrome critical region was selected (1100). PRIMER-BLAST (1102) was used to design 100 PCR primers (1104) in 50,000 bp regions. Unique sequences on chromosome 21 were sought to generate approximately 140-150 bp fragments. Primers were selected from different clusters in different regions on chromosome 21 (1106) and synthesized and arranged in 96 well plates (1108).

[0109]FIG. 12 illustrates a primer pair that was designed, indicating length, annealing position on chromosome 21, melting temperature (Tm), and percent GC content. FIG. 13 illustrates the positions of three 50 kbp regions in a Down syndrome critical region on chromosome 21. FIG. 14 illustrates Bioanalyzer results of PCR amplification of different sequences from clusters A, B, and C in regions A, B, and C on chromosome 21. FIG. 15 illustrates amplification results f...

example 3

Selection of Hotspot Region for Amplification

[0113]Sequences for enrichment can be chosen on the basis of being in a “hotspot,” a region of relatively high sequence coverage. FIG. 19 illustrates that sequence runs from multiple samples were combined to give 79% coverage of chromosome 21. The bottom chart illustrates Illumina pipeline output files containing multiple files and each given start and end chromosome positions; therefore the sequencing reads cover 37 M region (46,927,127 last position-9,757,475 1st position=˜37 M). FIG. 20 shows a schematic of chromosome 21 to which sequence reads have been mapped. Some regions have more sequence coverage than other regions. FIG. 21 illustrates an example of a process that was used to select a specific region of 251 base pairs for amplification. Sequence within 13,296,000-46,944,323 (illustrated in FIG. 20) was selected for amplification. FIGS. 22A and B illustrate the relative position for a Down syndrome critical region (35,892,000-41,7...

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Abstract

Methods and kits for selectively enriching non-random polynucleotide sequences are provided. Methods and kits for generating libraries of sequences are provided. Methods of using selectively enriched non-random polynucleotide sequences for detection of fetal aneuploidy are provided.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 297,755, filed Jan. 23, 2010, which application is incorporated herein by reference.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 May 11, 2011, is named 32477692.txt and is 27,793 bytes in size.BACKGROUND OF THE INVENTION[0003]Massively parallel sequencing techniques are used for detection of fetal aneuploidy from samples that comprise fetal and maternal nucleic acids. Fetal DNA often constitutes less than 10% of the total DNA in a sample, for example, a maternal cell-free plasma sample. Sequencing a large number of polynucleotides to generate sufficient data for fetal aneuploidy detection can be expensive. Methods for randomly enriching fetal nucleic acids in cell-free maternal sample have been described, including enriching...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C40B20/00
CPCC12Q1/6869C12Q2600/16C12Q2600/156C12Q1/6883C12Q2600/112
Inventor CHUU, YUE-JENRAVA, RICHARD P.
Owner VERINATA HEALTH INC
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