Boris Tichý

DNA re-sequencing

DNA re-sequencing
Comparisson with reference sequence (genome)

•
•
•
•
•

WGS – whole genome sequencing
WES – whole exome sequencing
Genes, gene panels
SNV – single nucleotide variant
CNV – copy number variant

CEITEC at Masaryk University

2

DNA re-sequencing
WGS – whole genome sequencing
• DNA sequencing without selection
• Fragmentation
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Ultrasound

–

Enzymes

Longer fragments (> 300bp)
• PCR-free – GC-bias
• Paired-end sekvencing
• Germline - 30x coverage ~ 100Gb
–

CEITEC at Masaryk University

3

DNA re-sequencing
WES – whole exome sequencing
• Coding sequence selection
• Fragmentation
–

Ultrasound

–

Enzymes

Fragments 150-250bp
• PCR
• Paired-end sequencing
–

CEITEC at Masaryk University

4

DNA re-sequencing
WES – whole exome sequencing
• Coding sequence selection
• Fragmentace
–

Ultrasound

–

Enzymes

Fragments 150-250bp
• PCR
• Paired-end sekvenace
• Germline - >50x coverage, >3Gb
–

CEITEC at Masaryk University

5

DNA re-sequencing
Gene panels
• Coding sequence selection
• Fragmentation
–

Ultrasound

–

Enzymes

Fragments 150-250bp
• PCR
• Paired-end sekvenace
• Germline - >50x coverage
–

CEITEC at Masaryk University

6

DNA re-sequencing
Gene panels
•
•
•
•
•
•
•

Coding sequence selection
PCR enrichment
Single primer PCR
Inverse PCR + capture
Capture + probe extension
Paired-end sequencing
Germinální - >50x coverage

CEITEC at Masaryk University

7

DNA re-sequencing
Gene panels
•
•
•
•
•
•
•

Coding sequence selection
PCR enrichment
Single primer extension
Inverzní PCR + capture
Capture + probe extension
Paired-end sekvenace
Germinální - >50x coverage

CEITEC at Masaryk University

8

DNA re-sequencing
Gene panels
•
•
•
•
•
•
•

Coding sequence selection
PCR enrichment
Single primer PCR
Inverse PCR + capture
Capture + probe extension
Paired-end sekvenace
Germinální - >50x coverage

CEITEC at Masaryk University

9

DNA re-sequencing
Gene panels
•
•
•
•
•
•
•

Coding sequence selection
PCR enrichment
Single primer PCR
Inverse PCR
Capture + probe extension
Paired-end sekvenace
Germinální - >50x coverage

CEITEC at Masaryk University

10

DNA re-sequencing
Somatic variants
• Tumor x Normal

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WGS, WES, panels

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Tumor only variants

Tumor only
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Panels, genes

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Variants germline i somatic

Interpretation according to VAF, anotations, ...
• UMI, Duplex-seq adaptors
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Identification of PCR duplicates

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Error correction

CEITEC at Masaryk University

11

CEITEC
@CEITEC_Brno

Thank you
for your attention
www.ceitec.eu