Sequencing Technology Flashcards
Understanding the laboratory technique and scientific basis behind different kinds of sequencing.
ChIP-qCR assay
Chromatin immunoprecipitation, or ChIP, uses Abs to selectively enrich specific DNA-binding proteins and their DNA targets. ChIP can investigate a particular protein-DNA interaction, several protein-DNA interactions, or interactions across the whole genome or a subset of genes.
ChIP utilizes antibodies that selectively recognize and bind histones, histone modifications, transcription factors, or cofactors, to provide information about chromatin states and gene transcription. Used to understand gene expression and regulation in cells or tissues of interest.
Cytogenetics
Study of tissue, blood, bone marrow or cultured cells to look for changes in chromosomes, such as broken, missing, rearranged or extra chromosomes.
ddPCR
Droplet Digital PCR. An emulsion PCR process that performs absolute quantitation by dividing nucleic acid samples into thousands of nanoliter-sized droplets. Each droplet provides an individual reaction. Better for samples with low concentration of nucleic acids or variable levels of chemical and protein contaminants than normal qPCR.
Differential gene expression analysis
A statistical analysis of normalized read-count data to discover quantitative changes in expression levels between groups.
FISH
Fluorescence in-situ hybridization (ISH). FISH uses fluorescently labeled nucleic acid (DNA or RNA) probes to bind to specific target sequences within cells or tissue samples and is quicker and more sensitive than ISH. FISH uses fluorescent microscopy and can multiplex.
Flow cytometry
Measures the number of cells, the percentage of live cells, and certain characteristics of cells (size, presence of antigens, shape) in a sample. The cells are stained with a light-sensitive dye, placed in a fluid, and then passed one at a time through a beam of light. The measurements are based on how the stained cells react to the beam of light. Most often used for leukemia, lymphoma and PNH.
HLA typing
Blood-based NGS human leukocyte antigens (HLA) typing. Identifies surface major histocompatibility complexes (MHC). Used by oncologists, doctors assessing patients for transplant. Performed via flow cytometry or NGS for more detailed results.
Immunohistochemistry
A laboratory method that uses antibodies to check for certain antigens (markers) in a sample of tissue. The antibodies are usually linked to an enzyme or a fluorescent dye. These stains are used to detect the presence, relative quantity and localization of specific proteins to aid in determining differentiation in neoplasms with similar morphology. In addition to other applications, IHC is used to provide prognostic or therapeutic information.
ISH
In-situ hybridization. ISH is slower and less sensitive than FISH. Uses using a non-fluorescent hapten labeled strand of DNA, RNA, or modified nucleic acids to detect sequences within intact cells or preserved tissue sections. Uses immunohistology.
Liquid biopsy molecular profile
Detects circulating extracellular nucleic acids such as cfDNA (cell-free DNA), ctDNA (circulating tumor DNA) and CTCs (circulating tumor cells) from blood or other bodily fluids.
Methylation sequencing
Provides insight into gene regulation. DNA methylation may also be used as a biomarker. Used in addition to NGS to provide additional context. Done via methylation microarray, such as Illumina’s Infinium assay. Needs a large amount of DNA from heterogeneous cells, which may be difficult to collect (the case with circulating tumor cells).
Microarray
A multiplex “lab on a chip” used to perform genotyping. An array of nucleic acid fragments are bound to a solid surface, then bathed in genetic material. Pairing between the sample and the chip-immobilized fragments generated fluorescence, which is used to measure gene expression or detect SNPs. Illumina uses a bead-based microarray, called BeadChip. Can profile several thousand genes at a time. Bulk method, like qPCR. Need to know genes in advance, validation tool.
Mitochondrial (mtDNA) sequencing
Provides insight into mitochondrial disorders. Catches point mutations and deletions, heteroplasmy (more than one mtDNA sequence variant per sample). Can be done via whole-exome sequencing, or targeted sequencing. Targeted sequencing, for example, may be limited to analysis of the D-loop hypervariable region, a noncoding region of mtDNA where many genetic alterations may occur.
PGx
Pharmacogenetic testing. Uses NGS to identify genes associated with adverse reactions to certain pharmaceuticals. Can identify toxicity, lack of efficacy, or hypersensitivity.
qPCR
Quantitative real-time PCR. Unlike conventional PCR, it uses fluorescent signal intensities to measure relative and absolute amounts of DNA present (gene expression). Usually used for a single gene, but it can be multiplexed for multiple targets. Needs pre-designed gene-specific primers (no discovery). Bulk method, really only for a few genes at a time.
Real-time PCR
PCR that monitors amplification of target during PCR, as opposed to afterwards (conventional PCR).
RNASeq
NGS as applied to RNA. Fulgent performs whole exome/genome sequencing with RISE analysis, which targets coding regions and splice junctions. Allows for discovery. Bulk method.
RT-PCR
Reverse transcription PCR for RNA detection and quantification. Still “real-time” PCR.
Sanger sequencing
“Chain termination method” of sequencing. Now typically automated, though originally done by hand. PCR is performed with fluorescent, chain terminating ddNTPs. DNA fragments are then separated by size via gel electrophoresis. A laser is used to excite the fluorescent ddNTPs, and the resulting fluorescence is detected by a sequencing machine.
scATACseq
Single cell assay for transposase-accessible chromatin using sequencing (ATAC-seq). Detects chromatin landscape associated with a given cell type. Doesn’t require advance knowledge of epigenetic marks or transcription factors. Helps ID cell type and state, epigentic modifications. Needs less cells than other tests.
Solid tumor molecular profile
Lumera NGS. Tests a sample of a solid tumor for genetic abnormalities via DNA assay. May be paired with an RNA-based assay as well. See liquid tumor biopsy. Small sample size needed.
Tapestri comprehensive single-celled DNA panels (AML, CLL Myeloid)
Flexible single cell-based assay for cancer researchers in biopharma. Uses cell lines, PMBC, BMMC. Very customizable — can use DNA, protein or multi -omics panels. Identifies SNVs (single nucleotide variant), indels, CNVs, LOH (loss of heterozygosity) and translocations. Not fully validated in DNA + protein multiplexed panel.
Visium CytAssist Spatial Gene Expression
Maps transcriptome within context of FFPE and fresh frozen tissue. Works via slides with barcoded spots, each of which has many capture probes. Tissue placed on the slide releases RNA, which binds to probes. Barcoded cDNA is made from captured RNA; this is then captured off the slide and analyzed spatially and fed into a standard sequencer.
Whole-exome sequencing (WES)
Includes all coding DNA. Can expand to untranslated regions and microRNA to view gene regulation. Produces less data and may be lower cost.
