Diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing

Abstract

Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

1 - 23 . (canceled) 
     
     
         24 . A method for determining a presence or absence of a fetal aneuploidy in a fetus for each of a plurality of maternal blood samples obtained from a plurality of different pregnant women, said maternal blood samples comprising fetal and maternal cell-free genomic DNA, said method comprising:
 (a) obtaining a fetal and maternal cell-free genomic DNA sample from each of the plurality of maternal blood samples;   (b) selectively enriching a plurality of non-random polynucleotide sequences of each fetal and maternal cell-free genomic DNA sample of (a) to generate a library derived from each fetal and maternal cell-free genomic DNA sample of enriched and indexed fetal and maternal non-random polynucleotide sequences, wherein each library of enriched and indexed fetal and maternal non-random polynucleotide sequences includes an indexing nucleotide sequence which identifies a maternal blood sample of the plurality of maternal blood samples,   wherein said plurality of non-random polynucleotide sequences comprises at least 100 different non-random polynucleotide sequences selected from a first chromosome tested for being aneuploid and at least 100 different non-random polynucleotide sequences selected from a reference chromosome, wherein the first chromosome tested for being aneuploid and the reference chromosome are different, and wherein each of said plurality of non-random polynucleotide sequences is from 10 to 1000 nucleotide bases in length,   (c) pooling the libraries generated in (b) to produce a pool of enriched and indexed fetal and maternal non-random polynucleotide sequences;   (d) performing massively parallel sequencing of the pool of enriched and indexed fetal and maternal non-random polynucleotide sequences of (c) to produce sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences of each of the at least 100 different non-random polynucleotide sequences selected from the first chromosome tested for being aneuploid and sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences of each of the at least 100 different non-random polynucleotide sequences selected from the reference chromosome;   (e) based on the indexing nucleotide sequence, for each of the plurality of maternal blood samples, enumerating sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences selected from the first chromosome tested for being aneuploid and sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences selected from the reference chromosome; and   (f) for each of the plurality of maternal blood samples, determining the presence or absence of a fetal aneuploidy comprising using a number of enumerated sequence reads corresponding to the first chromosome and a number of enumerated sequence reads corresponding to the reference chromosome of (e).   
     
     
         25 . The method of  claim 24 , wherein for each of the plurality of maternal blood samples determining the presence or absence of a fetal aneuploidy comprises comparing the number of enumerated sequence reads corresponding to the first chromosome tested for being aneuploid with the number of enumerated sequence reads corresponding to the reference chromosome. 
     
     
         26 . The method of  claim 24 , wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from the first chromosome tested for being aneuploid and at least 300 different non-random polynucleotide sequences selected from the reference chromosome. 
     
     
         27 . The method of  claim 26 , wherein said plurality of non-random polynucleotide sequences comprises at least 500 different non-random polynucleotide sequences selected from the first chromosome tested for being aneuploid and at least 500 different non-random polynucleotide sequences selected from the reference chromosome. 
     
     
         28 . The method of  claim 24 , wherein each of said plurality of non-random polynucleotide sequences is from 10 to 500 nucleotide bases in length. 
     
     
         29 . The method of  claim 24 , wherein each of said plurality of non-random polynucleotide sequences is from 50 to 150 nucleotide bases in length. 
     
     
         30 . The method of  claim 24 , wherein said first chromosome tested for being aneuploid is selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y. 
     
     
         31 . The method of  claim 30 , wherein said fetal aneuploidy comprises fetal aneuploidy of a chromosome selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y. 
     
     
         32 . The method of  claim 31 , wherein said fetal aneuploidy is selected from the group consisting of trisomy 21, trisomy 18, trisomy 13, and monosomy X. 
     
     
         33 . The method of  claim 24 , wherein said reference chromosome is selected from the group consisting of chromosome 1, chromosome 2, chromosome 3, chromosome 13, chromosome 18, and chromosome 21. 
     
     
         34 . The method of  claim 24 , wherein said fetal aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of the first chromosome. 
     
     
         35 . The method of  claim 24 , wherein said selectively enriching of (b) comprises performing polymerase chain reaction (PCR) amplification. 
     
     
         36 . The method of  claim 35 , wherein for each fetal and maternal cell-free genomic DNA sample PCR amplification comprises hybridizing at least two oligonucleotides to each of the at least 100 different non-random polynucleotide sequences selected from the first chromosome tested for being aneuploid and each of the at least 100 different non-random polynucleotide sequences selected from the reference chromosome. 
     
     
         37 . The method of  claim 36 , wherein said oligonucleotides do not hybridize to non-random polynucleotide sequences comprising one or more polymorphisms. 
     
     
         38 . The method of  claim 36 , wherein each of said oligonucleotides has a substantially similar melting temperature. 
     
     
         39 . The method of  claim 24 , wherein said massively parallel sequencing generates at least 30 nucleotide bases per sequence read. 
     
     
         40 . The method of  claim 24 , wherein said fetal aneuploidy comprises partial monosomy or partial trisomy. 
     
     
         41 . The method of  claim 24 , wherein said plurality of non-random polynucleotide sequences comprises no more than 1000 different non-random polynucleotide sequences selected from the first chromosome tested for being aneuploid and no more than 1000 different non-random polynucleotide sequences selected from the reference chromosome. 
     
     
         42 . A method for determining a presence or absence of a fetal aneuploidy in a fetus for each of a plurality of maternal blood samples obtained from a plurality of different pregnant women, said maternal blood samples comprising fetal and maternal cell-free genomic DNA, said method comprising:
 (a) obtaining a fetal and maternal cell-free genomic DNA sample from each of the plurality of maternal blood samples;   (b) selectively enriching a plurality of non-random polynucleotide sequences of each fetal and maternal cell-free genomic DNA sample of (a) to generate a library derived from each fetal and maternal cell-free genomic DNA sample of enriched and indexed fetal and maternal non-random polynucleotide sequences, wherein each library of enriched and indexed fetal and maternal non-random polynucleotide sequences includes an indexing nucleotide sequence which identifies a maternal blood sample of the plurality of maternal blood samples,   wherein said plurality of non-random polynucleotide sequences comprises at least 100 different non-random polynucleotide sequences selected from at least one chromosome region tested for being aneuploid and at least 100 different non-random polynucleotide sequences selected from at least one chromosome control region, wherein the at least one chromosome region tested for being aneuploid and the at least one chromosome control region are different, and wherein each of said plurality of non-random polynucleotide sequences is from 10 to 1000 nucleotide bases in length;   (c) pooling the libraries generated in (b) to produce a pool of enriched and indexed fetal and maternal non-random polynucleotide sequences;   (d) performing massively parallel sequencing of the pool of enriched and indexed fetal and maternal non-random polynucleotide sequences of (c) to produce sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences of each of the at least 100 different non-random polynucleotide sequences selected from the at least one chromosome region tested for being aneuploid and sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences of each of the at least 100 different non-random polynucleotide sequences selected from the at least one chromosome control region;   (e) based on the indexing nucleotide sequence, for each of the plurality of maternal blood samples, enumerating sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences selected from the at least one chromosome region tested for being aneuploid and sequence reads corresponding to enriched and indexed fetal and maternal non-random polynucleotide sequences selected from the at least one chromosome control region; and   (f) for each of the plurality of maternal blood samples, determining the presence or absence of a fetal aneuploidy comprising using a number of enumerated sequence reads corresponding to the at least one chromosome region tested for being aneuploid and a number of enumerated sequence reads corresponding to the at least one chromosome control region of (e).   
     
     
         43 . The method of  claim 42 , wherein for each of the plurality of maternal blood samples determining the presence or absence of a fetal aneuploidy comprises comparing the number of enumerated sequence reads corresponding to the at least one chromosome region tested for being aneuploid with the number of enumerated sequence reads corresponding to the at least one chromosome control region. 
     
     
         44 . The method of  claim 42 , wherein said plurality of non-random polynucleotide sequences comprises at least 300 different non-random polynucleotide sequences selected from the at least one chromosome region tested for being aneuploid and at least 300 different non-random polynucleotide sequences selected from the at least one chromosome control region. 
     
     
         45 . The method of  claim 42 , wherein each of said plurality of non-random polynucleotide sequences is from 10 to 500 nucleotide bases in length. 
     
     
         46 . The method of  claim 42 , wherein the at least one chromosome region tested for being aneuploid is selected from at least one chromosome selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y. 
     
     
         47 . The method of  claim 46 , wherein said fetal aneuploidy comprises fetal aneuploidy of a chromosome selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y. 
     
     
         48 . The method of  claim 47 , wherein said fetal aneuploidy is selected from the group consisting of trisomy 21, trisomy 18, trisomy 13, and monosomy X. 
     
     
         49 . The method of  claim 42 , wherein the at least one chromosome control region is selected from at least one chromosome selected from the group consisting of chromosome 1, chromosome 2, chromosome 3, chromosome 13, chromosome 18, and chromosome 21. 
     
     
         50 . The method of  claim 42 , wherein said fetal aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of at least one chromosome. 
     
     
         51 . The method of  claim 42 , wherein said selectively enriching of (b) comprises performing polymerase chain reaction (PCR) amplification. 
     
     
         52 . The method of  claim 51 , wherein for each fetal and maternal cell-free genomic DNA sample PCR amplification comprises hybridizing at least two oligonucleotides to each of the at least 100 different non-random polynucleotide sequences selected from the at least one chromosome region tested for being aneuploid and each of the 100 different non-random polynucleotide sequences selected from the at least one chromosome control region. 
     
     
         53 . The method of  claim 42 , wherein said plurality of non-random polynucleotide sequences comprises no more than 1000 different non-random polynucleotide sequences selected from the at least one chromosome region tested for being aneuploid and no more than 1000 different non-random polynucleotide sequences selected from the at least one chromosome control region.