PYROSEQUENCINGNucleic acid sequencing by synthesis

Sam Okewumi

Objective

oIntroduction

oSanger Sequencing

oPyrosequencing

Instrumentation, preparation and results

oApplication

oAdvantage and Disadvantage

oSo What?

WHAT IS DNA SEQUENCING?

Process of determining the sequencing of the 4 nucleotides in nucleic acid molecule

o Gives an insight into genetic organizationo Blueprint for understanding the genetic function of an organismo Sequence organism that cannot be cultured “microbial dark matter”

WHY SEQUENCE?

theconversation.com: Beethoven’s Ninth Symphony

researchgate.com: Typical DNA sequence

HISTORY OF DNA SEQUENCING

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DISCOVERY

OF DNA

DNA PROPOSED AS

“GENETIC MATERIAL”

DOUBLE HELIX

DNA STRUCTURE

SANGER

SEQUENCING

PYROSEQUENCING

COMPLETE

SEQUENCE OF

H. influenzae

I WAS BORN

DRAFT OF

HUMAN GENOMEA LOT

SANGER SEQUENCING: 1st GEN

o Single stranded DNA template

o A primer for DNA synthesis

o DNA polymerase

o Deoxynucleoside triphosphates and dideoxynucleotide triphosphates

o Fluorescence or radioactive labelled

Anneal the primer, polymerase extends, visualize fragments on gel

electropherogram

Sequencing by chain termination

What is needed?

Results

Visualize pattern on gel and sequence is detected by computer to generate a electropherogram

Khan Academy: DNA sequencing ddNTP

AtdBio.com: Sanger Sequencing

Harrington, C., Lin, E., Olson, M., & Eshleman, J. (2013). Fundamentals of pyrosequencing.(Report). Archives of Pathology & Laboratory Medicine, 137(9), 1296–1303. https://doi.org/10.5858/arpa.2012-0463-RA

Is there any method for large scale sequencing?

Advantages

High sensitivity and AccuracyCapable of large scale sequencingLong individual read length (400-900 bp)

Limitations

Sequence one DNA molecule at a timeExpensive and impractical for large sequencing projects

SANGER SEQUENCING: 1st GEN

PYROSEQUENCING:

Giphy.com: Austin Powers

PYROSEQUENCINGSequencing by direct synthesis using DNA polymerase

Pyrophosphate (PPi) is release upon the incorporation of a nucleotide by DNA polymerase

Cascade of reactionsreleased PPi is converted to ATP by sulfurylaseATP provides energy for luciferase to oxidize luciferin and emit a photon of light

Flashes of light and their intensity measured by a detector and recorded

Washing step to remove excess substrate after each nucleotide addition

Principle

**dATP nucleotide substituted with dATPαS due to being a substrate for luciferasedATPas is inert for luciferase but is still effectively incorporated by DNA polymerase

Researchgate: Structure of dATPαS

Nature.com: Pyro Q-CpGTM

Harrington, C., Lin, E., Olson, M., & Eshleman, J. (2013). Fundamentals of pyrosequencing.(Report). Archives of Pathology & Laboratory Medicine, 137(9), 1296–1303. https://doi.org/10.5858/arpa.2012-0463-RA

INSTRUMENTATION

o Automated system delivers small volume into microtiter plate

Moves in the X-Y direction

o Individual nucleotides are dispensed into the wells according to the specified order

Microtiter plate continuously agitated as nucleotide added to ensure mixing

o CCD camera images the microtiter plates every second to follow the reaction

o Data System controls instrument and dispensation order of the nucleotide

o Software allows for analysis of selected wells

Schematicscolar.org: Pyrosequencing Schematic

Schematicscolar.org: microtiter plate

Schematicscolar.org: flowgram from single well

Harrington, C., Lin, E., Olson, M., & Eshleman, J. (2013). Fundamentals of pyrosequencing.(Report). Archives of Pathology & Laboratory Medicine, 137(9), 1296–1303. https://doi.org/10.5858/arpa.2012-0463-RA

PREPARATIONo Fragmentation of DNAo Attach adapters to both ends of the DNAo Denature ds-DNA to single strando Incubate with the solid support beads

beads coated with streptavidincontain primer that links to one of the adaptor

o PCR amplify DNA on beadcycle of elongating denaturing annealing until whole bead is covered with same DNA

o Washing and filtrationremove left over nt, fragments that didn’t attach or bead with different fragments

o Transfer beads into the wells on microtiter plate size of well only allows 1 bead per wellplate contains all the reagents other than the nucleotides

Schematicscolar.org: pyrosequencing workflow

Random DNA Fragment A1A2

Harrington, C., Lin, E., Olson, M., & Eshleman, J. (2013). Fundamentals of pyrosequencing.(Report). Archives of Pathology & Laboratory Medicine, 137(9), 1296–1303. https://doi.org/10.5858/arpa.2012-0463-RA

SEQUENCING

o Primer complementary to adaptor sequence is added

o Nucleotide bases are added to the wells one at a time in a timed fashion

o If nucleotide is incorporated light signal produced and recorded by the cameraintensity of light corresponds to number of nucleotide added by polymerase

o Wash with apyrase to remove left over nucleotide

o Add the next nucleotide

Schematicscolar.org: pyrosequencing workflow

Harrington, C., Lin, E., Olson, M., & Eshleman, J. (2013). Fundamentals of pyrosequencing.(Report). Archives of Pathology & Laboratory Medicine, 137(9), 1296–1303. https://doi.org/10.5858/arpa.2012-0463-RA

RESULTS

Height (intensity) of peak indicates the number of dNTPs added

This sequence: GCAGGCCT

Software

Simplified Example

ATATGGGTCCTAAGTGGCGTGCTTTTTGATGCTTC

TCAAAGGGAACAATTCCCCTCTAGAAATAATTTTG

TTTAACTTTAAGAAGGAGATATACATATGCACCAC

CACCACCACCACCCCATGGGTATGAATAAGCAAAA

GGTTTGTCCTGCTTGTGAATCTGCGGAACTTATTT

ATGATCCAGAAAGGGGGGAAATAGTCTGTGCCAAG

TGCGGTTATGTAATAGAAGAGAACATAATTGATAT

GGGTCCTAAGTGGCGTGCTTTTG

Sequence output

Researchgate.com: pyrosequencing output

JoVe.com: Pyrosequencing for Microbial Identification and Characterization

TRANSLATE THE SEQUENCE

WHAT TO DO WITH DATA?o Look for ORF or genes

promotor region, RB site (SD sequence)

o Compare to other genomeso use BLAST

search database for similar sequencetranslate to amino acid sequence and characterize protein function

o Characterize genotype and phenotype

o Analyze sequencepredict genes, mRNA/regulatory RNA and proteinprograms such as artemis “genome browser and annotation tool”

o DATABASE : NCBIupload sequence to GenBank

APPLICATION

SpringerLink.com: Pyrosequencing application

Marsh, S. (2007). Pyrosequencing applications. Methods in Molecular Biology (Clifton, N.J.), 373, 15–24.

ADVANTAGES *thumbs up emoji*

o Rapid analysis, high throughputo Thousands of sequence can be obtained in single runo Cost per nucleotide much lower when compared to sanger sequencingo Easily automatedo No need for separation on a gel

DIS-ADVANTAGES *thumbs down emoji*

o Detecting homopolymer (repeating nucleotides) regions due to nonlinearity of light responseo Shorter read length 400-500 baseso expensive instrument/equipment/software

Would generally be found in routine laboratories that perform large quantity of sequencing

Harrington, C., Lin, E., Olson, M., & Eshleman, J. (2013). Fundamentals of pyrosequencing.(Report). Archives of Pathology & Laboratory Medicine, 137(9), 1296–1303. https://doi.org/10.5858/arpa.2012-0463-RA

oMiniaturize the technique

oDecrease cost

oLonger reads

oLess error

Future Trends

Giphy.com: Kanye Kobe

o Improve capability

SEQUENING PROJECTS

GEBA project

American Gut project

o Launched by the US National institute of Health (NIH)o Characterize diversity of the microbiome in the human body

For more info: http://humanfoodproject.com/americangut/

o “Genomic Encyclopedia of Bacteria and Archaea”o Project launched by the JGI (Joint Genome Institute)o Aim is to fill in gaps in the bacterial and archaeal branches of the tree of life

For more info: https://jgi.doe.gov/further-expansion-of-the-geba-project/

Kyrpides, N., Hugenholtz, P., Eisen, J., Woyke, T., Göker, M., Parker, C., … Chun, J. (2014). Genomic Encyclopedia of Bacteria and Archaea: Sequencing a Myriad of Type Strains. 12(8), e1001920. https://doi.org/10.1371/journal.pbio.1001920

EXTRA

Round Up

“... [A] knowledge of sequences could contribute much to our understanding of living matter.”-Frederick Sanger

Frederick Sanger – Biographical. NobelPrize.org. Nobel Media AB 2020. Fri. 28 Feb 2020. <https://www.nobelprize.org/prizes/chemistry/1980/sanger/biographical/>

o Genome sequencing has been a remarkable benefit to biological research

o Knowledge of sequence can be used for many things

Find genes

screen for characteristic features of genes

discover novel genes

comparative purposes

increase phylogenetic diversity

better understand organism

“A LOT”

ohttps://www.dna-worldwide.com/resource/160/history-dna-timeline

oMILESTONESo Jay Shendure, Shankar Balasubramanian, George M. Church, Walter Gilbert,

Jane Rogers, Jeffery A. Schloss, & Robert H. Waterston. (2017). DNA sequencing at 40: past, present and future. Nature, 550(7676), 345–353. https://doi.org/10.1038/nature24286

oNEW SEQUENCING METHODSoHeather JM, Chain B. The sequence of sequencers: The history of sequencing

DNA. Genomics. 2016;107(1):1–8. doi:10.1016/j.ygeno.2015.11.003

ohttps://www.youtube.com/watch?v=jFCD8Q6qSTM