Sanger Sequencing Validation
Sanger Sequencing Validation - Related Videos
01:57
Sanger Sequencing
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2021
DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
07:17
Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
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Cited by 1 •
2024
This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.
04:58
Introductory Analysis and Validation of CUT&RUN Sequencing Data
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Cited by 1 •
2024
This protocol guides bioinformatics beginners through an introductory CUT&RUN analysis pipeline that enables users to complete an initial analysis and validation of CUT&RUN sequencing data. Completing the analysis steps described here, combined with downstream peak annotation, will allow users to draw mechanistic insights into chromatin regulation.
05:45
Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example
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Cited by 15 •
2020
We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.
01:29
Reliability and Validity
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2020
Reliability and validity are two important considerations that must be made with any type of data collection. Reliability refers to the ability to consistently produce a given result. In the context of psychological research, this would mean that any instruments or tools used to collect data do so in consistent, reproducible ways. Unfortunately, being consistent in measurement does not necessarily mean that you have measured something correctly. To illustrate this concept, consider a kitchen...
FAQs
When incorporated by DNA polymerase, each fluorescently labeled dideoxynucleotide terminates the growing DNA strand at a particular base. Because the reaction produces fragments ending at different positions, the collection records the sequence across the targeted region. This termination pattern converts nucleotide incorporation into an ordered fluorescent signal that can be read after fragment separation.
Capillary electrophoresis separates the terminated fragments according to their lengths, while their fluorescent labels identify the terminal bases. The resulting chromatogram presents the targeted sequence as an ordered pattern of signals. Comparing that pattern with a reference sequence allows investigators to determine whether the observed nucleotide change is present and whether the readout agrees with the expected sequence.
Assessment of zygosity depends on how the observed sequence pattern relates to the reference at the variant position. The chromatogram supplies the experimental readout, and the reference provides the expected comparison point. This pairing helps distinguish a confirmed nucleotide change from a possible sequencing error, making the result more useful for interpreting a molecular finding.
It provides orthogonal evidence, meaning an independent sequencing readout for the same suspected finding. The targeted reaction focuses on the specific genetic variant rather than relying only on the original result. Agreement between the targeted sequence and the reference can strengthen confidence in the variant before it is considered in a molecular diagnosis.
The workflow begins with a DNA template and a primer directed at the region of interest. DNA polymerase extends that primer in the presence of fluorescently labeled dideoxynucleotides, generating terminated fragments. Capillary electrophoresis then separates the fragments, and the resulting chromatogram is compared with a reference sequence. Interpretation addresses the suspected nucleotide change, zygosity, and possible errors.
It is most relevant when a specific genetic variant requires confirmation for accurate clinical interpretation. The method can verify a finding generated by next-generation sequencing and provide additional evidence supporting a molecular diagnosis. Sanger sequencing validation can also be applied to engineered constructs, although that use belongs to molecular research rather than clinical interpretation.
For an engineered construct, the generated sequence can be compared with a reference representing the intended design. Agreement supports the presence of the expected nucleotide sequence, while a discrepancy identifies a sequence that requires further consideration. This application uses the same targeted readout and reference-based comparison that support variant confirmation in medical investigations.