CLIA Whole Genome Sequencing and Whole Exome Sequencing
Whole genome sequencing requires an extremely high amount of sequencing throughput to generate a moderate depth of coverage. The data generated, while comprehensive, does not allow detection of mutations with as much sensitivity as a targeted approach. Exome sequencing is the most cost-effective and efficient solution.
A large portion of relevant mutations occur in the exome. In fact, the exome contains as many as 85% of disease-related mutations. Covering less than 2% of the whole genome, exome sequencing requires only 1/50th of the sequencing throughput to generate the same depth of coverage. This approach provides flexible experimental options:
- Maintain the same depth of coverage and multiplex more samples into the same sequencing lane, significantly decreasing total project cost
- Increase the depth of coverage to facilitate the detection of rare, low-frequency mutation
- Germline/frequent variants: 50-100x
- Somatic/rare variants: ≥200x
- Tumor vs Normal: ≥200x tumor, ≥100x normal
- Population studies: 50-100x
CLIA PCR and Sanger Sequencing
Choose “CLIA Workflow” if you need CLIA lab, CLIA personnel, CLIA workflow, QA oversight, assay performed in triplicate at the assay development stage, no test registration, no clinical outcome reporting, and lab director signature to ensure CLIA-compliant sequencing. This uses the workflow and activities of the validated CLIA test but does not use a registered test from the test roster.
Choose “CLIA” if you need CLIA lab, CLIA personnel, CLIA workflow, QA oversight, assay validation (to assess accuracy, sensitivity, reproducibility), test registration, clinical outcome reporting, and lab director signature.
Click here for a visual summary of CLIA Environment, CLIA Workflow, and CLIA full service.
Have a specific question?
Email | Phone 1-877-GENEWIZ 436-3949, Ext. 3350
CLIA Sanger Sequencing
Whole genome sequencing requires an extremely high amount of sequencing throughput to generate a moderate depth of coverage. The data generated, while comprehensive, does not allow detection of mutations with as much sensitivity as a targeted approach. Exome sequencing is the most cost-effective and efficient solution.
A large portion of relevant mutations occur in the exome. In fact, the exome contains as many as 85% of disease-related mutations. Covering less than 2% of the whole genome, exome sequencing requires only 1/50th of the sequencing throughput to generate the same depth of coverage. This approach provides flexible experimental options:
- Maintain the same depth of coverage and multiplex more samples into the same sequencing lane, significantly decreasing total project cost
- Increase the depth of coverage to facilitate the detection of rare, low-frequency mutation
- Germline/frequent variants: 50-100x
- Somatic/rare variants: ≥200x
- Tumor vs Normal: ≥200x tumor, ≥100x normal
- Population studies: 50-100x
Pre-Defined: Your sample concentration has been adjusted following our guidelines and you are supplying your primer in a separate tube for GENEWIZ to add (additional charges apply).
Custom: GENEWIZ will purify your PCR template and add your primers (additional charges apply). Please note that Plasmid DNA samples do not qualify for this service option.
CLIA Integration Site Analysis (ISA)
| Sample Type | Quantity |
| Cells | 1 million |
| gDNA | > 500 ng |
| Fresh frozen tissues | 10-30 mg |
We offer two analytically validated NGS-based assays, leveraging targeted approaches to identify, quantify, and monitor viral vector integration events across the genome. Both assays generate information on insertion site location and frequency as well as offer the following unique capabilities:
Target Enrichment Sequencing (TES)
A Hybridization-Capture TES Assay that generates information on insertion site location and frequency, and additionally allows for insertion/transgene integrity study, which is important to resolve mutations or rearrangements.
Quantitative Shearing Linear Amplification Mediated-PCR (qsLAM-PCR)
A Targeted qsLAM-PCR Sequencing Assay that focuses on identifying insertion site location and frequency, offering superior sensitivity on insertion site detection.
Both of our analytically validated NGS-based assays are efficient at generating information about where the viral vector has inserted into the host genome:
Linear Amplification-Mediated-PCR (LAM-PCR) focuses on insertion site location and frequency, offering superior sensitivity on insertion site detection;
Hybrid Capture Targeted Enrichment Sequencing (TES) generates information on insertion site location and frequency, and additionally allows for insertion/transgene integrity study, which is important to resolve mutations or rearrangements.
Lentiviral vectors are efficient gene delivery vehicles suitable for delivering long-term transgene expression in various cell types over time. A drawback of the lentiviral vector include the risk of insertional mutagenesis due to the semi-random integration of genes.
Guidance from the FDA and other regulatory authorities, is that patients treated with cell or gene therapies utilizing viral vectors be monitored for 5 – 15 years for safety. This monitoring includes, but not limited to, vector integration studies of patient samples to ensure stability.
Per the FDA, “LTFU observations are extended assessments that continue some of the scheduled observations of a clinical trial past the active follow-up period and are an integral portion of the study of some investigational GT products. LTFU observations are important to monitor long term safety of GT products. For GT products that present long term risks to subjects, LTFU/surveillance plan(s) should also be put in place post-licensure for monitoring of delayed adverse events (for details we refer you to section VI. of this document).”
Guidelines from the FDA suggest 15 years of long-term follow up after gene therapy treatment with integrating vectors such as lentiviral vectors. Analysis should be performed to determine the site of vector integration if the analysis of a subject’s surrogate cells suggests a predominant clone (e.g., oligoclonal pattern of vector insertions) or monoclonality. In addition, if you detect a predominant integration site, test for persistence by performing another analysis for clonality no more than three months later.
1) Insertion site location, frequency, abundance
2) Integration hotspots & CPG islands
3) Oncogenic gene annotation of Integration Site
4) Longtitudinal profiling (clonal expansion)
5) Vector integrity (TES)
Custom analysis support options are also available upon consultation.
Microarray Solutions
- ThermoFisher manufactures microarrays by synthesizing targets in situ. Arrays are offered in a 96-well plate and may contain 800,000+ targets in each well. Microarrays are scanned on a ThermoFisher GeneTitan instrument with analysis using the Axiom Analysis Suite Software. ThermoFisher offers microarrays for genotyping and gene expression analysis.
- Illumina utilizes silica microbeads adorned with hundreds of thousands to millions of genotypes. These beads are housed in etched microwells and coated with multiple copies of an oligonucleotide probe targeting a specific genomic locus. The beads contain one extra base which enables incorporation of a labeled nucleotide, which can be detected on Illumina’s iScan instrument, and analyzed using GenomeStudio software. Illumina offers bead chip arrays for genotyping or methylation analysis.
- For ThermoFisher Axiom arrays, 96 samples are genotyped per plate. For ThermoFisher GO Screen (gene expression) arrays, 384 samples are processed per plate.
- For Illumina bead chip arrays, the number of samples depends on the bead chip array of interest. Typically, these are processed as 8, 16, 24, 32, or 96 samples.
- ThermoFisher microarrays: Both positive (HapMap) and negative (no template) controls, provided by ThermoFisher, are used in the Axiom (genotyping) arrays. For a ThermoFisher Axiom array (96-wells), only 94 samples are available for customer samples as 2 positions are required for these controls. For ThermoFisher GO Screen (384-wells), one NTC and one positive control are used per plate.
- Illumina bead chips: A single positive control, provided by Illumina, is used per run, regardless of the number of bead chips in the run.
