Maternal plasma transcriptome analysis by massively parallel RNA sequencing

A method of diagnosing a pregnancy-associated disorder using a sample from a female subject pregnant with a fetus, the method comprising:

• applying an assay to RNA molecules obtained from the sample to generate a plurality of reads that include different alleles at one or more loci for measuring relative expression levels between the different alleles in the sample, wherein the assay does not target a reference gene for normalizing expression levels;
• receiving the plurality of reads, wherein the reads are obtained from an analysis of RNA molecules obtained from the sample, the sample containing a mixture of maternal- and fetal-derived RNA molecules;
• identifying, by a computer system, locations of the reads in a reference sequence;
• identifying one or more informative loci, each of which is homozygous in a first entity for a corresponding first allele and which is heterozygous in a second entity for the corresponding first allele and a corresponding second allele, wherein the first entity is the pregnant female subject or the fetus, and the second entity is the other one of the pregnant female subject and the fetus;
• filtering the one or more informative loci to identify one or more filtered informative loci:
• that are located within an expressed region of the reference sequence,
• at which at least a first predetermined number of reads in the plurality of reads containing the corresponding first allele are located, and
• at which at least a second predetermined number of reads in the plurality of reads containing the corresponding second allele are located,
• for each of the one or more filtered informative loci:
• determining a first number of reads located at the filtered informative locus and containing the corresponding first allele, and
• determining a second number of reads located at the filtered informative locus and containing the corresponding second allele,
• calculating a first sum of the first numbers and the second sum of the second numbers;
• calculating a ratio of the first sum and the second sum, and
• comparing the ratio to a cutoff value to determine whether the fetus, mother, or pregnancy has a pregnancy-associated disorder, the cutoff value being determined from one or more samples obtained from pregnant female subjects without the pregnancy-associated disorder, thereby determining whether the fetus, mother, or pregnancy has a pregnancy-associated disorder without normalizing with respect to a reference gene or a total amount of RNA molecules in the sample.