Exome Sequencing Analysis with Mitochondrial Analysis

Exome Sequencing Analysis with Mitochondrial Analysis
WSLH Department: Cytogenetics
WSLH Test Code:895M40
Includes:Whole Exome Sequencing (WES) is used to detect variants in protein-coding DNA. The exome will be sequenced to an average read depth of 85-100X. Over 97% of the exome will be fully covered at ≥10X read depth. The mitochondrial genome of the patient will be sequenced to a minimum read depth of 20X.
Methodology:Genomic DNA is isolated and the exonic regions and flanking splice junctions are captured and enriched using Agilent SureSelect V5. Mitochondrial DNA is isolated and amplified by long-range PCR. Exome and mitochondrial sequencing libraries are combined and undergo massively parallel sequencing using the Illumina HiSeq 2500 sequencing system with 100 base pair (bp) paired-end reads. DNA sequences are aligned and compared to reference gene sequences based on human genome build GRCH37/UCSC hg19. A custom bioinformatics analysis pipeline is used to compare sequence changes (variants) in the proband to the reference sequence. Variants with a minimum coverage of 10X are analyzed. Variant annotation and sorting is performed using Cartagenia Bench software. Each variant is evaluated using databases (e.g., ClinVar, 1000 Genomes, NHLBI GO Exome Sequencing Project, etc), published literature, clinical correlation, segregation analysis, and predicted functional or splicing impact (using computational tools such as PolyPhen, SIFT, etc). Variant classification follows ACMG guidelines (PMID:25741868). Interpretations may change over time as more information becomes available. If parental samples are available at the time of submission, segregation analysis may be performed to inform variant interpretation. All reported sequence variants are confirmed by Sanger sequencing or an appropriate alternate method. Depth of coverage may be reported for oncology/somatic variant evaluation specimens. Mitochondrial analysis has been assessed to detect a minimum of 20% heteroplasmy. A portion of this test is run in UWCS facility CLIA#52D2089533 425 Henry Mall, Madison, WI 53706.
Turn-around Time:100 Days
Recommended Uses: 
Specimen Requirements:Adults and children: 5 mL (minimum) to 10 mL (preferred) whole blood collected in EDTA (purple-top) tube(s). *Infants (0-1 year): 2 mL of whole blood collected in EDTA (purple-top) tube(s). Submission of parental specimens (biological mother and father) is recommended.
Collection Kit/Container: 
Patient Preparation: 
Collection Instructions: 
Specimen Handling and Transport:Store and transport specimens at room temperature (may transport with coolant during hot, >85 degrees F weather). DO NOT FREEZE. The laboratory must receive specimens within 24-48 hours of collection.
Unacceptable Conditions:Blood that is clotted or hemolyzed is not acceptable. Blood must not be frozen. Plasma and serum are not acceptable.
Requisition Form: 
Required Information:Laboratory regulations require the following minimum information to be provided on the requisition form for a specimen to be accepted for testing: Patient name or unique identifier; date and time of collection, patient date of birth and sex, specimen type/site of collection, test request(s), reason for referral (detailed phenotype information), clinician name and UPIN/NPI, and address for reporting results. Please be certain that name/identifier on the form matches that on the specimen label.
Results include: 
Limitations:This assay is designed to cover protein coding regions only and variants that lie within introns and untranslated regions may not be detected. At this time, whole exome sequencing cannot detect single and multi-exon deletions or duplications. In addition, large genomic rearrangements may not be detected unless breakpoints occur within the sequenced region. Trinucleotide repeat expansions and epigenetic changes such as DNA methylation are not evaluated. Whole exome sequencing does not provide complete coverage of all coding exons.
Additional Tests Recommended:SNP array CGH is recommended for the detection of copy number variations.
Additional Comments: 
Additional Tests Performed: 

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