Microbiology - Sequencing techniques

14 important questions on Microbiology - Sequencing techniques

What is an example of first generation sequencing?

Sanger sequencing.

What is sanger sequencing?

A chain-termination method that enables determination of DNA base order and can be used to sequence a complete genome.

How is the DNA sequence identified in Sanger sequencing?

A fluorescence detector identifies the label attached to the ddNTP and the end of each DNA fragment.
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What do the fluorescent signals represent?

The nucleotide at the end of the terminated fragment.

What is an example of Second generation sequencing?

Next Generation Sequencing.

How does NGS work?

It is based on selective incorporation of chain-terminating dideoxynucleotides during DNA replication.

Why are short DNA fragments used for Illumina sequencing?

Because longer fragments are not stable enough to stand straight.

What is an example of third generation sequencing?

Nanopore sequencing.

What are key metrics in choosing which sequencing method to use?

Read length, throughput, accuracy, cost and sample requirements.

For what is sanger sequencing often used as a benchmark?

For confirmation of genetic variations found with NGS.

What are strengths of short read sequencing?

It has highly accurate results, is fast and more affordable.

What are limitations of short read sequencing?

It can't sequence long DNA sequences directly, amplification can introduce errors or sequence biases and it has difficulties with complex genomic regions.

What are strengths of long read sequencing?

It improves accuracy of complex genomic regions and variants in repetitive or polymorphic regions.

How has the reduction in sequencing costs impacted research and clinical applications?

It has revolutionized the diagnosis and treatment of rare diseases and leads to earlier and more accurate diagnosis and personalized treatment.

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