Methods of fetal abnormality detection

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.

1 . (canceled) 
     
     
         2 . A method for determining the presence or absence of fetal aneuploidy in a maternal blood sample, the method comprising:
 (a) obtaining a maternal blood sample comprising fetal and maternal cell-free DNA;   (b) selectively enriching a plurality of non-random polynucleotide sequences of genomic DNA from cell-free DNA in a maternal blood sample to generate a library of enriched non-random polynucleotide sequences found in said fetal and maternal cell-free DNA, wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from a chromosome tested for being aneuploid, said enriching comprising:
 (i) a first amplification step to generate a plurality of first reaction products, said amplification comprising at least 300 first primers configured to amplify at least 300 different non-random polynucleotide sequences, wherein each of said plurality of non-random polynucleotide sequences is from 10 to 500 nucleotide bases in length; 
 (ii) a second amplification step to generate second reaction products, said amplification comprising a second set of primers comprising sequences contained in the first reaction products; and 
 (iii) a third amplification step to generate a third reaction product comprising said library of enriched non-random polynucleotide sequences, said amplification comprising a third set of primers comprising sequences contained in the second reaction products; and
 wherein at least one primer of at least one of the second and third sets of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to permit the enriched non-random polynucleotide sequences of the library to anneal to a same sequencing primer for the enriched non-random polynucleotide sequences of the library; 
 
   (c) sequencing said enriched non-random polynucleotide sequences;   (d) enumerating sequence reads from said sequencing step; and   (e) determining the presence or absence of fetal aneuploidy based on said enumerating.   
     
     
         3 . The method of  claim 2 , wherein at least one primer of the third set of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to add an index to the enriched non-random polynucleotide sequences of the library, wherein the index indicates the maternal blood sample from which the library was generated. 
     
     
         4 . The method of  claim 2 , wherein primers of the second set of primers contain universal sequences such that nucleic acids of the second reaction product contain common universal sequences added to the different non-random polynucleotide sequences, and wherein the third set of primers are configured to hybridize at least in part to the common universal sequences. 
     
     
         5 . The method of  claim 4 , wherein at least one primer of the third set of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to add an index to the enriched non-random polynucleotide sequences of the library, wherein the index indicates the maternal blood sample from which the library was generated. 
     
     
         6 . The method of  claim 2 , wherein at least one primer of at least one of the second and third sets of primers includes a sequence configured to be added to the different non-random polynucleotide sequences to add an index to the enriched non-random polynucleotide sequences of the library, wherein the index indicates the maternal blood sample from which the library was generated. 
     
     
         7 . The method of  claim 2 , wherein said plurality of non-random polynucleotide sequences comprises at least 500 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid. 
     
     
         8 . The method of  claim 2 , wherein each of said plurality of non-random polynucleotide sequences is from 10 to 260 nucleotide bases in length. 
     
     
         9 . The method of  claim 2 , wherein each of said plurality of non-random polynucleotide sequences is from 50 to 150 nucleotide bases in length. 
     
     
         10 . The method of  claim 2 , wherein said selectively enriching comprises performing PCR. 
     
     
         11 . The method of  claim 2 , wherein said selectively enriching comprises linear amplification. 
     
     
         12 . The method of  claim 2 , wherein said non-random polynucleotide sequences comprise sequences that are sequenced at a rate of greater than 5-fold more than other sequences on the chromosome. 
     
     
         13 . The method of  claim 2 , wherein said chromosome tested is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y. 
     
     
         14 . A method for determining the presence or absence of fetal aneuploidy in a maternal blood sample, the method comprising:
 (a) obtaining a maternal blood sample comprising fetal and maternal cell-free DNA;   (b) selectively enriching a plurality of non-random polynucleotide sequences of genomic DNA from cell-free DNA in a maternal blood sample to generate a library of enriched non-random polynucleotide sequences found in said fetal and maternal cell-free DNA, wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from a chromosome tested for being aneuploid, wherein said selectively enriching comprises:
 (i) amplifying said plurality of non-random polynucleotide sequences from said maternal and fetal genomic DNA using a first pair of primers to form a first product, wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from a chromosome tested for being aneuploid, wherein each of said plurality of non-random polynucleotide sequences is from 10 to 500 nucleotide bases in length; 
 (ii) amplifying the first product of (i) with a second set of primers comprising sequences contained in the first product of (i) to form a second product; and 
 (iii) amplifying the second product of (ii) with a third set of primers comprising sequences contained in the second product of (ii);
 wherein one of said second or third sets of primers includes an indexing sequence; 
 
   (c) sequencing said enriched non-random polynucleotide sequences;   (d) enumerating sequence reads from said sequencing step; and   (e) determining the presence or absence of fetal aneuploidy based on said enumerating.   
     
     
         15 . The method of  claim 14 , wherein said third set of primers includes the indexing sequence. 
     
     
         16 . The method of  claim 14 , wherein said indexing sequence distinguishes polynucleotides in the maternal blood sample from polynucleotides in a different maternal blood sample. 
     
     
         17 . The method of  claim 14 , wherein said cell-free DNA is from a plurality of maternal blood samples from a plurality of different individuals, and wherein said indexing sequence identifies a maternal blood sample from each of said plurality of different individuals. 
     
     
         18 . The method of  claim 14 , wherein said plurality of non-random polynucleotide sequences comprises at least 400 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid. 
     
     
         19 . The method of  claim 14 , wherein said plurality of non-random polynucleotide sequences comprises at least 500 different non-random polynucleotide sequences selected from the chromosome tested for being aneuploid. 
     
     
         20 . The method of  claim 14 , wherein each of said plurality of non-random polynucleotide sequences is from 10 to 260 nucleotide bases in length. 
     
     
         21 . The method of  claim 14 , wherein each of said plurality of non-random polynucleotide sequences is from 50 to 150 nucleotide bases in length. 
     
     
         22 . The method of  claim 14 , wherein said selectively enriching comprises performing a polymerase chain reaction (PCR). 
     
     
         23 . The method of  claim 14 , wherein said selectively enriching comprises linear amplification. 
     
     
         24 . The method of  claim 14 , wherein said chromosome tested is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.