NGS: Nucleic acid from the submitted specimen is enriched for coding and adjacent noncoding regions of the genes on the panel using a custom capture kit (ArcherDx, Boulder, CO). The library products were sequenced with 2 by 150 bp reads on either the Illumina Miseq or NextSeq sequencing instruments (Illumina, San Diego, CA). After alignment to the reference genome (hg19), off target, low quality and duplicate reads are removed and variants are detected with the variant calling algorithms in the software listed below. This test detects single nucleotide substitutions (SNVs), small insertions and deletions, and copy number variations located in the DNA coding sequences and known splice regions in the genes targeted by the panel. All sequence alterations are described according to the Human Genome Variation Society (HGVS) nomenclature guidelines and are classified according to the guidelines for sequence variant interpretation of the American College of Medical Genetics and Genomics (ACMG). Variant classification categories include pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign, and benign with VUS, likely benign and benign variants excluded from the report.

Spinal Muscular Atrophy (SMA): SMA status is assessed via copy number analysis using the data generated via the NGS panel described above. Normalized coverage of exons 7-8 are compared against healthy cohort data to determine copy status. SMN1 specific primers are part of the NGS panel and are used to detect the silent allele polymorphism (g.27134T>G).

Fragile X: A PCR-based assay is used to determine the size of the CGG repeat region of FMR1. The reported number of repeats is estimated to be accurate within two repeats for the normal and intermediate ranges and five repeats for the premutation to full mutation.