Sequencing: Hybridization capture-based and amplification-based NGS

Hybridization capture-based enrichment uses chemically synthesized nucleic acid probes that are complementary to and specifically bind target DNA fragments. This allows efficient physical separation of the target DNA from the rest of the genome. Hybridization capture enables high-throughput testing of relatively large target regions (dozens to thousands of genes) and yields exceptional performance for molecular oncology and constitutional disease testing, including high accuracy on archived formalin-fixed specimens. Additionally, this approach allows for the detection of certain rearrangements and, in some cases, copy number alterations, in addition to single nucleotide variants (SNVs) and small insertions and deletions (indels).

Amplification-based enrichment employs traditional polymerase chain reaction (PCR) to enzymatically generate many copies of specific target DNA sequences. These PCR products, referred to as “amplicons”, can be loaded directly onto a next-generation sequencing platform like the Illumina HiSeq. While this technique can be moderately cheaper and faster than hybridization capture-based methods, it is limited to detecting only SNVs and indels, usually across significantly smaller target regions.

Microarray AND qPCR: SNP genotyping CNV and expression analysis

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Cytogenetics: Karyotyping, FISH and chromosomal microarray

A variety of clinically validated, routine cytogenetic tests may be ordered through Washington University Pathology Services and are briefly described below. For more information on how cytogenetic technologies can be incorporated into customized workflows for clinical research and trials, please contact us.

Karyotyping is the classical cytogenetic analysis used to identify numerical and structural abnormalities at the chromosome level. Routine karyotype analysis is most often performed by GTG-banding (Giemsa staining), however analysis using C or NOR banding is also available. Karyotype analysis used in the diagnosis of congenital disorders is validated for a number of specimen types including amniotic fluid, chorionic villi, products of conception, and blood.

Fluorescence in situ hybridization (FISH) is used to establish the diagnosis, determine the classification, and monitor the treatment of a vast number of congenital and acquired chromosome abnormalities. FISH analysis can be performed on prenatal and neonatal specimens for the detection of congenital disorders, and oncology specimens for the detection of inherited and acquired malignancies.

Validated specimens for prenatal and postnatal analysis include amniotic fluid, chorionic villi, peripheral blood, and products of conception. FISH analysis performed on these specimens includes the early and rapid detection of trisomies and microdeletions which constitute a large number of congenital disorders.

Validated specimen types for the diagnosis and monitoring of many types of cancer include bone marrow, whole blood, and solid tissue specimens. FISH analysis performed on these specimens includes detection of translocations, deletions, duplications, amplifications, and monitor the status of bone marrow transplants. These chromosomal abnormalities are characteristic of a large number of hematological and solid tumor disorders and malignancies.

Chromosomal microarray analysis (CMA) is a high resolution molecular genetic test which is able to detect very small gains and losses across the entire genome that are not seen by more traditional karyotype or FISH tests.  CMA testing is performed for a wide range of constitutional disorders, prenatal testing, products of conception (POC) and cancer. Common constitutional indications are developmental delay, neurologic or neuromuscular abnormalities, multiple congenital anomalies, dysmorphic features and cardiac abnormalities. Cancer CMA testing is offered for hematological malignancies and solid tumors.

Bioninformatics: Sequence analysis, annotation and interpretation tools

Bioinformatics at Washington University School of Medicine (WUSM) is a highly collaborative and synergistic enterprise between the Bioinformatics Core and the Center for Biomedical Informatics (CBMI). These groups bring complementary skills and interests in the areas of software development and analysis together to serve the diverse needs of WUSM clinical and research faculty and our collaborators.

The bioinformatics team has been responsible for remarkable innovation in the area of clinical genomics, building the Clinical Genomicist Workstation (CGW) to manage all aspects of clinical next-generation sequencing-based testing. CGW offers a HIPAA-compliant cloud-based bioinformatics solution that tracks and analyzes patient specimens from the time of accessioning through DNA isolation and sequencing, genomic alignment of sequence results and clinical interpretations based on a custom clinical annotation database.

The CGW software application was recently licensed and is now available through its Washington University-sponsored distributing entity, PierianDX.