US 12,270,073 B2
Methods for preparing a biological sample obtained from an individual for use in a genetic testing assay
Matthew Rabinowitz, San Francisco, CA (US); George Gemelos, Portland, OR (US); Milena Banjevic, Los Altos Hills, CA (US); Allison Ryan, Belmont, CA (US); Zachary Demko, San Francisco, CA (US); Matthew Hill, Belmont, CA (US); Bernhard Zimmermann, Manteca, CA (US); Johan Baner, San Francisco, CA (US); and Styrmir Sigurjonsson, San Jose, CA (US)
Assigned to Natera, Inc., San Carlos, CA (US)
Filed by Natera, Inc., San Carlos, CA (US)
Filed on Feb. 20, 2020, as Appl. No. 16/795,973.
Application 16/795,973 is a continuation of application No. 16/399,991, filed on Apr. 30, 2019.
Application 16/399,991 is a continuation of application No. 14/532,666, filed on Nov. 4, 2014.
Application 14/532,666 is a continuation of application No. 13/791,397, filed on Mar. 8, 2013, granted, now 9,163,282, issued on Oct. 20, 2015.
Application 13/791,397 is a continuation of application No. 13/300,235, filed on Nov. 18, 2011, granted, now 10,017,812, issued on Jul. 10, 2018.
Application 13/300,235 is a continuation in part of application No. 13/110,685, filed on May 18, 2011, granted, now 8,825,412, issued on Sep. 2, 2014.
Claims priority of provisional application 61/542,508, filed on Oct. 3, 2011.
Claims priority of provisional application 61/571,248, filed on Jun. 23, 2011.
Claims priority of provisional application 61/516,996, filed on Apr. 12, 2011.
Claims priority of provisional application 61/448,547, filed on Mar. 2, 2011.
Claims priority of provisional application 61/462,972, filed on Feb. 9, 2011.
Claims priority of provisional application 61/398,159, filed on Jun. 21, 2010.
Claims priority of provisional application 61/395,850, filed on May 18, 2010.
Prior Publication US 2020/0181697 A1, Jun. 11, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12P 19/34 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6862 (2018.01); C12Q 1/6869 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6883 (2018.01); G01N 33/48 (2006.01); G16B 20/00 (2019.01); G16B 20/10 (2019.01); G16B 20/20 (2019.01); G16B 20/40 (2019.01); C12N 15/11 (2006.01); G16B 40/00 (2019.01)
CPC G16B 20/00 (2019.02) [C12Q 1/6806 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6862 (2013.01); C12Q 1/6869 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6883 (2013.01); G16B 20/10 (2019.02); G16B 20/20 (2019.02); G16B 20/40 (2019.02); C12N 15/11 (2013.01); C12Q 2525/179 (2013.01); C12Q 2527/113 (2013.01); C12Q 2527/143 (2013.01); C12Q 2537/143 (2013.01); C12Q 2537/149 (2013.01); C12Q 2537/159 (2013.01); C12Q 2545/114 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/16 (2013.01); G16B 40/00 (2019.02)] 27 Claims
OG exemplary drawing
 
1. A method for preparing a biological sample obtained from a single individual for use in a genetic testing assay, the method comprising:
identifying 50 to 10000 polymorphic loci for targeting DNA of the single individual in the biological sample;
isolating cell-free DNA fragments from the biologic sample, wherein the isolated DNA fragments comprise DNA from the 50 to 10000 targeted polymorphic loci;
ligating the isolated DNA fragments with a plurality of adaptors comprising a first known tail sequence, and a molecular barcode to produce adaptor-ligated DNA fragments such that most or all of the adaptor-ligated DNA fragments comprising a same targeted locus have a unique molecular barcode, wherein the first known tail sequence comprises a first universal priming sequence;
amplifying at least some of the adaptor-ligated DNA fragments comprising DNA from the 50 to 10000 targeted polymorphic loci using a universal primer that binds to the first universal priming sequence and 50 to 10000 different target-specific primers in one PCR reaction volume, followed by a second, nested PCR amplification, to obtain amplified adaptor-ligated DNA fragments comprising the first known tail sequence, a molecular barcode, and a target sequence; and
sequencing at least some of the amplified DNA fragments by clonal sequencing to generate allelic data for the 50 to 10000 targeted polymorphic loci.