Genomic Test Methods
HNL Genomics offers Next Generation Sequencing (NGS) panels which provide high throughput sequencing of many genes simultaneously and is useful for disorders associated with multiple genes.
Next Generation Sequencing for Inherited Genetics
- This method offers 100% coverage of targeted exons and exon boundaries, for the detection of insertions and deletions, and complete selectivity in the presence of pseudogenes. ·
- Exons that are not completely covered by at least 40 NGS reads are sequenced using Sanger sequencing. Additionally, all potentially pathogenic sequence variants identified by NGS are confirmed by Sanger sequencing. Similar to Sanger sequencing, this method will detect point mutations, splice site mutations, and small insertions and deletions. ·
- HNL Genomics remains actively involved in evaluating new methodologies for mutation detection. These methodologies will be considered for introduction if they are proven to be reliable, accurate and superior or complementary to existing technology.
- For NGS Testing, the turnaround time is based on the type of test being performed and can be found in the test profiles in our genomics test directory.
Targets are amplified by PCR and sequenced using ABI3730 sequencers. All potentially pathogenic variants are confirmed by additional Sanger sequencing. Sanger sequencing is considered the gold standard for mutation detection and is highly sensitive for the detection of point mutations, splice site mutations, and small insertions and deletions.
Fragment analysis via Capillary Electrophoresis (CE) is a technique in which fluorescently labeled PCR DNA fragments are separated by size, with the ability to generate relative quantitation of DNA fragments and genotyping information.
Fluorescently labeled primers are used to amplify targeted DNA region(s) during PCR. The PCR products containing any amplified fluorescent DNA fragments are then injected into long narrow capillaries filled with polymer. A high voltage is applied causing the negatively charged DNA fragments to migrate toward the positive end with larger fragments migrating more slowly through the capillary. The size separated fluorescent DNA fragments are then detected by a laser/camera system and size compared to an internal standard. Fragments differing by only one base pair can be accurately sized. Analysis by peak intensity measurements provides relative quantitation information.
For Fragment Analysis via Capillary Electrophoresis, the turnaround time is based on the type of test being performed and can be found in the test profiles in our test directory.
Fluorescence in situ hybridization (FISH) can detect specific sites of specific DNA sequences in metaphase or interphase cells. FISH results are used for diagnosis, prognosis, for predicting the response to therapy and for surveillance. FISH involves unwinding of the double helix structure and binding of the DNA of all probes attached to a fluorescent molecule with a specific sequence of sample DNA, which can be visualized under the fluorescent microscope.
There are different types of probes available based on the targeted region:
- whole chromosomal regions (chromosome painting)
Some examples of diseases diagnosed using FISH include:
- Angelman syndrome
- Prader-Willi syndrome
- Trisomies 13, 16, 18, 21
- Monosomy X
- Turner syndrome
- Different types of leukemia/lymphomas and solid tumors.
- bone marrow
- peripheral blood
- amniotic fluid
- lymph nodes
The advantage of FISH testing is the short Turnaround time. The specimens are analyzed using MetaSystems. FISH can reveal a lot about a tumor that help make the treatment successful but must be specific for a change.
Chromosome analysis is a type of genetic testing that looks at the size, shape, and number of chromosomes in a sample of cells from your body, aiding in the diagnosis of genetic diseases, some birth defects, and certain disorders of the blood and lymphatic system.
Chromosome analysis is performed when pregnancy screening tests are abnormal; whenever signs of a chromosomal abnormality-associated disorder are present; as indicated to detect chromosomal abnormalities in a person and/or detect a specific abnormality in family members; sometimes when a person has leukemia, lymphoma, myeloma, myelodysplasia or another cancer and an acquired chromosome abnormality is suspected. The sample is cultured based on the diagnosis followed by harvesting, slide dropping, staining and analysis using MetaSystems and interpretation.
- bone marrow
- lymph node
- amniotic fluid
- fresh tissues
Single nucleotide polymorphism (SNP) Array provides the ability to identify genomic copy number losses and copy number gains at a resolution that is much higher than the traditional chromosomal karyotype analysis and FISH. SNP microarray is the hybridization of fragmented single-stranded DNA to arrays containing hundreds of thousands of unique nucleotide probe sequences. Specialized equipment is then used to produce a measure of the signal intensity associated with each probe and its target after hybridization. The signal intensity depends upon the amount of target DNA in the sample, as well as the affinity between target and probe.
SNP microarray is recommended as a first-tier genetic test for the postnatal evaluation of individuals with:
- multiple congenital anomalies
- developmental delay
- intellectual disability
- autism spectrum disorders
SNP array helps in detecting genomic gains and losses including:
- unbalanced translocations
- copy neutral aberrations like loss of heterozygosity (LOH) / Absence of heterozygosity (AOH), and >20% mosaicism.
It cannot detect balanced translocations, Robertsonian, Balanced insertions, Inversions, <20% culture mosaicism, chromosomal position of genomic gains. The samples include bone marrow, peripheral blood, amniotic fluid or tissues.