10-SeqTechLabProtocols.Rmd

# Sequencing Technologies and Lab Protocols

In order to understand any sequencing technology, you have to understand basic chemistry of nucleic acids:

![Boris (PNG), SVG by Sjef, Public domain, via Wikimedia Commons](./Figures/DNAchem.png){width=100%}

A DNA sequence is a string of As, Gs, Ts, and Cs. This string is synthesized by the formation of phosphodiester bonds between nucleotides (the sugar-phosphate backbone).

Which of the following is DNA and which is RNA?

![](./Figures/DNARNA.png){width=80%}

Here we see thymidine triphosphate being integrated into a strand of DNA. What happens between the OH and the triphosphate?

![](./Figures/DNARep.png){width=60%}


NTP, dNTP, ddNTP

![](./Figures/NTPs.png){width=60%}

So what do you think happens if there isn't an oxygen available at the 3' location?

![](./Figures/Halt.png){width=60%}


## Sequencing technologies

#### Short Reads{-}

  + Sanger
  + Illumina
    
#### Long Reads{-}

  + PacBio
  + Oxford Nanopore
  
### Sanger Sequencing

Each reaction has very small amounts of ddNTPs.

  + 1 type of ddNTP per reaction
  + all reactions have the same DNA
  
![](./Figures/Sanger1.png){width=80%}

Four Separate Reactions

![](./Figures/Sanger2.png){width=80%}

Run the reactions on a gel to get the different sizes (lengths) of DNA per nucleotide. 

![](./Figures/Sanger3.png){width=80%}

What is the sequence of the following gel?

![](./Figures/Sanger4.png){width=80%}

Fluorescent ddNTPs:

![Fibonachi, CC BY-SA 1.0 <https://creativecommons.org/licenses/by-sa/1.0>, via Wikimedia Commons](./Figures/FloNTPs.png){width=60%}

If colors are assigned to GATC from above:

![](./Figures/Sanger5.png){width=60%}


Fluorescent peaks of nucleotides:

![Abizar at English Wikipedia, CC BY-SA 3.0 <http://creativecommons.org/licenses/by-sa/3.0/>, via Wikimedia Commons](./Figures/GelSeq.png){width=30%}


The order of sequences determined by the lengths at which they terminated at a specific ddNTP.

![](./Figures/Sanger6.png){width=60%}

In summary, the Sanger Sequencing process looks like this:

![Estevezj, CC BY-SA 3.0 <https://creativecommons.org/licenses/by-sa/3.0>, via Wikimedia Commons](./Figures/SangerSum.png){width=100%}

## Illumina Sequencing

![](./Figures/Illumina1.png){width=60%}

![](./Figures/Illumina2.png){width=60%}

![](./Figures/Illumina3.png){width=90%}

![](./Figures/Illumina4.png){width=60%}

https://www.youtube.com/watch?v=womKfikWlxM&t=80s

![](./Figures/Illumina5.png){width=100%}

![](./Figures/Illumina6.png){width=100%}

![](./Figures/Illumina7.png){width=100%}

https://www.youtube.com/watch?v=fCd6B5HRaZ8


## PacBio Sequencing

![](./Figures/Pac1.png){width=60%}

https://www.youtube.com/watch?v=_lD8JyAbwEo


![](./Figures/Pac2.png){width=60%}

https://www.youtube.com/watch?v=NHCJ8PtYCFc

![](./Figures/Pac3.png){width=60%}

![](./Figures/Pac4.png){width=60%}

![](./Figures/Pac5.png){width=60%}

![](./Figures/Pac6.png){width=60%}

![](./Figures/Pac7.png){width=60%}

![](./Figures/Pac8.png){width=60%}

![](./Figures/Pac9.png){width=60%}

![](./Figures/Pac10.png){width=60%}

https://www.youtube.com/watch?v=xbpYvki4U64


## Oxford Nanopore Sequencing

![](./Figures/ON1.png){width=80%}

https://www.youtube.com/watch?v=CGWZvHIi3i0


![](./Figures/ON2.png){width=60%}

![](./Figures/ON3.png){width=60%}

![](./Figures/ON4.png){width=80%}

https://www.youtube.com/watch?v=GUb1TZvMWsw


![](./Figures/ON5.png){width=100%}