Limitations of genomic sequencing

Exome limitations

The whole exome sequence test and associated Sanger sequencing confirmation is designed to evaluate single nucleotide variants within the human exome.  Using this technology it is only possible to sequence 90-95% of the human reference exome to the minimum 10-fold coverage estimated to be required to reliably detect heterozygous variants. The ability to identify abnormal variants is dependent on the presence of these sequence variants in the sequencing data. In addition, certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, long repeat sequences, triplet repeat expansions, structural chromosomal rearrangements, polyploidy, repetitive regions such as mono-, di- and tri-nucleotide repeats, GC rich regions, intronic variants outside of the splice-site, and epigenetic effects.  The test is limited in its ability to detect mosaicism. This technology is limited in its ability to accurately identify variants occurring in regions with high sequence identity to other regions of the genome (e.g. paralogous genes and pseudogenes).    

Interpretation assumes that any family relationships stated on the sample submission form are accurate. Secondary/incidental findings are routinely confirmed by Sanger sequencing in the proband only, but can be provided upon request for the parents (applies to trio analysis).

Variants found in a patient that are benign based on the medical literature, have an allele frequency greater than or equal to 1%, result in a synonymous amino acid change or occur in 5’ or 3’ untranslated regions are generally not reported, but may be made available on request.

Normal findings do not rule out the diagnosis of genetic abnormalities. If specific clinical disorders are suspected, specific evaluation of known genes by alternate test methods should be considered. Variants that interfere with DNA sequencing and medical procedures such as bone marrow transplantation and blood transfusion may result in misleading results.  The clinical implications of certain variants may be unknown at the time of analysis.  At the present time, the laboratory does not report variants found in the mitochondrial genome.  

Whole Genome Limitations