Sequencing Technologies: Illumina BIG BIO Juan De la Hoz THANKS - - PowerPoint PPT Presentation

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Sequencing Technologies: Illumina BIG BIO Juan De la Hoz THANKS - - PowerPoint PPT Presentation

Feb 11, 2023 157 likes •686 views

Sequencing Technologies: Illumina BIG BIO Juan De la Hoz THANKS BIG BIO What is Life? What is Life? What is Life? Reproduction Cell division DNA Replication What is Life? Reproduction Cell division DNA Replication

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BIG BIO

Sequencing Technologies: Illumina

Juan De la Hoz

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SLIDE 2

THANKS

BIO BIG

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What is Life?

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SLIDE 4

What is Life?

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SLIDE 5

What is Life?

Reproduction

  • Cell division

○ DNA Replication

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SLIDE 6

What is Life?

Reproduction

  • Cell division

○ DNA Replication

https://socratic.org/questions/how-does-dna-replication-relate-to-mitosis
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SLIDE 7

What is Life?

Reproduction

  • Cell division

○ DNA Replication

https://socratic.org/questions/how-does-dna-replication-relate-to-mitosis
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SLIDE 8

DNA Replication

https://byjus.com/biology/dna-replication-machinery-enzymes

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SLIDE 9

DNA Replication

https://byjus.com/biology/dna-replication-machinery-enzymes

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SLIDE 10

DNA Replication

https://byjus.com/biology/dna-replication-machinery-enzymes

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SLIDE 11

DNA Replication

https://byjus.com/biology/dna-replication-machinery-enzymes

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SLIDE 12

DNA Replication

https://byjus.com/biology/dna-replication-machinery-enzymes

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SLIDE 13

Sequencing by Synthesis

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Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

Imagine a small DNA molecule attached to a surface.

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Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

1. Add primers

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Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

1. Add primers 2. Add free nucleotides 3. Add polymerase

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Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

1. Add primers 2. Add free nucleotides 3. Add polymerase 4. Wash 5. Add light and take a photo

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SLIDE 18

Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

1. Add primers 2. Add free nucleotides 3. Add polymerase 4. Wash 5. Add light and take a photo 6. go back to 2

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SLIDE 19

Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

1. Add primers 2. Add free nucleotides 3. Add polymerase 4. Wash 5. Add light and take a photo 6. go back to 2

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SLIDE 20

Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

errors (1)

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SLIDE 21

DNA Replication

https://byjus.com/biology/dna-replication-machinery-enzymes

errors (1): Main driver of evolution

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Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

errors (1)

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SLIDE 23

Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing
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Seq by Synthesis

https://www.cegat.de/en/services/next-generation-sequencing

One big Advantage

  • Parallelization

One big Disadvantage

  • Short Reads

errors (2)

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SLIDE 25

PCR

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PCR

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

Signal Intensity can be Exponentially Amplified

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SLIDE 27

PCR

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

Signal Intensity can be Exponentially Amplified

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SLIDE 28

PCR

Adapters

https://www.mclab.com/DNA-Library-Prep-Kit.html
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SLIDE 29

PCR

Adapters

https://www.mclab.com/DNA-Library-Prep-Kit.html

Known Unknown Known

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PCR

https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced

Adapters Cluster formation

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PCR

https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced

Adapters Cluster formation

errors (1)

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SLIDE 32

PCR

https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced
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Fragment size (~500) Read length (150)

Single-end or Paired-end?

https://support.illumina.com/bulletins/2016/04/adapter-trimming-why-are-adapter-sequences-trimmed-from-only-the--ends-of-reads.html
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SLIDE 34

Library preparation

Genomic DNA Extraction Fragmentation Size selection Adapter ligation Attach to Plate Bridge PCR

RECAP

Sequencing by synthesis

Elongation (1nt) Fluorescence + Photo Repeat (paired-end) Invert molecule Repeat

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FASTQ FORMAT

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FASTQ FORMAT

. . . . . . . . .

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SLIDE 37

FASTQ FORMAT

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SLIDE 38

FASTQ FORMAT

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SLIDE 39

FASTQ FORMAT

Quality Score Encoding: Phred+33 (0-41)

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FASTQ FORMAT

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FASTQ FORMAT

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FASTQ FORMAT

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FASTQ FORMAT

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FASTQ FORMAT

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FASTQ FORMAT

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FASTQ FORMAT

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READ ALIGNMENT

https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

Reference Genome

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READ ALIGNMENT

https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

Reference Genome

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READ ALIGNMENT

https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

Reference Genome

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READ ALIGNMENT

https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

Reference Genome

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READ ALIGNMENT

https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

Reference Genome Coverage (1X, 10X, 30X, 100X)

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THANKS

BIO BIG