Multiplexed parallel analysis of targeted genomic regions for non-invasive prenatal testing

A method of testing for a risk of a chromosomal and/or subchromosomal abnormality in a sequencing library comprising a mixture of amplified maternal and fetal DNA, the method comprising:

• (a) preparing the sequencing library from the mixture of amplified maternal and fetal DNA and amplifying the sequencing library by polymerase chain reaction (PCR);
• (b) preparing custom synthetic Target Capture Sequences (TACS) by PCR or other DNA synthesis methods;
• (c) hybridizing the sequencing library to a pool of the TACS, wherein the pool comprises TACS that bind to one or more chromosomes or chromosomal regions of interest and wherein:
• (i) each TACS within the pool is between 150-260 base pairs in length, each TACS having a 5′ end and a 3′ end;
• (ii) each TACS within the pool binds to the chromosome(s) or chromosomal region(s) of interest at least 150 base pairs away, on both the 5′ end and the 3′ end, from regions harboring Copy Number Variations (CNVs), Segmental duplications or repetitive DNA elements;
• and
• (iii) the TACS have a GC content between 19% and 50%;
• (d) isolating members of the sequencing library that bind to the TACS to obtain an enriched library;
• (e) amplifying and sequencing the enriched library;
• (f) aligning, after sequencing, the enriched library to a human reference genome, thereby obtaining fragment-length information of the enriched library and a read-depth for one or more test loci from the enriched library and/or reference loci from the human reference genome;
• (g) performing multiple statistical analysis iteratively testing the read-depth for the one or more test loci against the read-depth for the one or more reference loci, thereby obtaining a component score, wherein the component score is obtained by:
• i. selecting a first random locus from a tested region;
• ii. recording a read-depth and GC content of the first random locus;
• iii. selecting a second random locus from a reference region, where the reference region is different from the tested region and wherein the second random locus has a GC content similar to the GC content of the first random locus;
• iv. recording a read-depth of the second random locus from the reference region, thereby providing a test-reference locus pair;
• v. repeating steps (i)-(iv) according to a total number of TACS in the tested region;
• vi. calculating a test score based on the difference in read-depth between each of the test-reference locus pairs from step (v);
• vii. repeating steps (i)-(vi) until a distribution of the test score converges to a stationary distribution;
• and
• viii. computing a first component risk score using the stationary distribution from step (vii);
• (h) performing a statistical analysis that groups fragments according to length and subsequently compares statistically a number of fragments in each group for the tested and reference regions to generate a second component risk score;
• and
• (i) assigning score values using multivariable method combining risk scores from step (g)(viii) and step (h), wherein the risk of the chromosomal and/or subchromosomal abnormality is classified as high, if the score values are above a reference threshold value.