Deletion/Duplication Analysis

Methodology

Sequence analysis

Covers all coding sequences and at least 30 base pairs of flanking intron sequence in the targeted genes at an average depth of 400 reads and a minimum depth of greater than 20 reads.

Deletion/duplication analysis

Detects intragenic deletions and duplications at single exon level resolution as well as whole gene deletions and duplications.  The bioinformatics method for copy number analysis is read depth calling which implies counting the number of reads in the targeted genomic interval, normalizing for total depth, and comparing to a reference set generated from samples known to be copy number neutral.

Our targeted NGS approach, using hybridization capture probes (Twist Bioscience), yields a high depth and uniformity of coverage across all coding exons and flanking intron sequences, which are prerequisites for reliable calling of copy number variants (CNVs).  We studied 125 samples with confirmed copy number status (previous aCGH), including 25 known CNVs of different sizes affecting different genes.  All known CNVs were detected by NGS, ranging from single exons to whole genes, with 100% concordance with aCGH.

The sensitivity of CNV calling is dependent on DNA quality.  The data quality will be highest if DNA was extracted on site from a fresh blood sample.

In cases of identified deletions or duplications:

• For intragenic deletion or duplication events, when technically feasible, we attempt to PCR amplify and Sanger sequence across the site for confirmation and to be able to offer targeted testing of relatives, if requested. When PCR amplification is not feasible, testing of relatives would require CNV analysis by NGS.
• For whole gene deletions or duplications, testing of relatives would require CNV analysis by NGS.
• For whole gene deletions or duplications, we may recommend whole genome array testing of the proband if the clinical phenotype is more complex than would be expected from altered copy number of a single gene. The Clinical Cytogenomics Laboratory in the Laboratory for Precision Diagnostics offers this testing (http://uwcpdx.org/constitutional-high-density-cytogenomic-microarray-analysis-cghsnp/).
• We will consider reporting incidental findings when the CNV may have a causal connection to the described clinical phenotype or when the CNV affects a gene in the list of medically actionable genes published by ACMG (Genet Med 23:1381-1390, 2021).  Before reporting, we will contact the referring health care provider to discuss the specific situation.

Specimen Requirements

Sample requirements for gene panels and single gene tests (includes deletion/duplication studies):

BLOOD: 2 EDTA (purple top) tubes
Adults: 5-10cc
Children: 3-5cc
Infants: 2-3cc

Whole blood may be stored up to 5-7 days in the refrigerator before shipping.

DNA:
5 µg DNA at a minimum concentration of ≥200 ng/µl.  DNA must be extracted in a CAP/CLIA or other suitably certified laboratory.

Special Instructions

In some instances, copy number variants are detected incidentally in targeted regions outside of the gene(s) of interest. The Collagen Diagnostic Laboratory makes every effort to alert ordering providers of incidental findings that are of known clinical significance and medically actionable. Please be aware that incidental findings may be detected regardless of the gene(s) ordered.

How to Order

Deletion/duplication studies are included as part of all new tests ordered at no additional charge.  At this time we cannot add deletion/duplication studies to a previous sequence-only test; testing must be run again using the new methodology.  To order these studies on a stored sample please complete a new Test Requisiton form (below) and fax it to the CDL at (206) 616-1899.

CDL Test Requisition Form

There is no additional charge for deletion/duplication studies, the prices currently shown for sequence analysis apply to both tests.