Genomic sequencing

There are two genomic tests, whole exome sequencing and whole genome sequencing:

Whole exome sequencing

Whole exome sequencing is performed by enriching for coding exons with target specific baits via a solution-based hybridization. Sequencing with an Illumina® HiSeq™ 2000 or 2500 is undertaken. The average exome wide depth of coverage is 100X. Whole exome sequencing determines the DNA sequencing of about 95% of the coding sequences and part of adjacent introns (non-coding sequences). This represents about 1.5% of the human genome and contains the part of the genome that is easiest to interpret. In our laboratory, a patient's exome is sequenced to an average depth of 100X.  Over 97% of the exome will be sequenced to a minimum depth of 10X.

Patient DNA sequence variants are loaded into a custom software package (Carpe Novo). Carpe Novo implements a number of algorithms and data sources allowing evaluation of whole exome sequencing variants for: evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites and presence in databases including OMIM, dbSNP, and HGMD. Carpe Novo annotates these variants with this data taking into account both the reference gene model and any identified alternate transcripts.

Variants of interest are confirmed by Sanger (dideoxy) sequencing. The DNA sequence of a segment of the genome surrounding each of these variants is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy sequencing.

Whole genome sequencing

Whole genome sequencing data is generated on Illumina sequencing instrumentation then DNA sequence variants are loaded into a custom software package (Carpe Novo). Carpe Novo implements a number of algorithms and data sources allowing evaluation of whole genome sequencing variants for: evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites and presence in databases including OMIM, dbSNP, and HGMD. Carpe Novo annotates these variants with this data taking into account both the reference gene model and any identified alternate transcripts. Whole genome sequencing determines the sequence of about 95% of the entire genome (coding and non-coding sequences) and is therefore a more complete and more expensive analysis than whole exome sequencing. In our laboratory, the patient's genome will be sequenced to an average depth of 40X. Over 90% of the genome will be sequenced to a minimum depth of 10X. The mitochondrial genome of the patient will be sequenced to a minimum depth of 20X.

Variants identified through whole genome sequencing are confirmed by Sanger sequencing. The DNA sequence of a segment of the genome surrounding each requested variant is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy sequencing.