Statistics Used in Final Scoring

For all chromosomes, paired-end sequencing data are aligned with the reference genome (HG19). Unique nonduplicated aligned reads are aggregated into 100 kb bins. The corresponding bin counts are adjusted for GC bias and according to previously established region-specific genomic coverage. Using such normalized bin counts, statistical scores are derived for each autosome by comparing the coverage regions that can be affected by aneuploidy with the rest of the autosomes. A log likelihood ratio (LLR) is computed for each sample by taking into account these coverage-based scores and the estimated FF. The LLR is the probability of a sample being affected given the observed coverage and FF versus the probability of a sample being unaffected given the same observed coverage. The calculation of this ratio also takes into account the estimated uncertainty in FF. For subsequent calculations, the natural logarithm of the ratio is used. The Assay software assesses the LLR for each target chromosome and each sample to provide a determination of aneuploidy.

Statistics for chromosomes X and Y are different from the statistics used for autosomes. For fetuses identified as female, SCA calls require classification agreement by LLR and by normalized chromosomal value.1Bianchi D, Platt L, Goldberg J et al. Genome‐Wide Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing. Obstet Gynecol. 2012;119(5):890–901. doi:10.1097/aog.0b013e31824fb482. Specific LLR scores are calculated for [45,X] (Turner syndrome) and for [47,XXX]. For fetuses identified as male, SCA calls for either [47,XXY] (Klinefelter syndrome) or [47,XYY] can be based on the relationship between the normalized chromosomal values for chromosomes X and Y (NCV_X and NCV_Y). Samples pertaining to male fetuses for which NCV_X is in the range observed for euploid female samples can be called [47,XXY]. Samples pertaining to male samples for which NCV_X is in the range observed for euploid male samples but for which chromosome Y is over represented can be called [47,XYY].

Some values of NCV_Y and NCV_X fall outside of the system's ability to make a determination of SCA. These samples produce a Not Reportable result for XY classification. Autosomal results are still provided for these samples if all other QC metrics are passed.