DNA re-sequencing
Boris Tichý
DNA re-sequencing
CEITEC at Masaryk University 2
• WGS – whole genome sequencing
• WES – whole exome sequencing
• Genes, gene panels
• SNV – single nucleotide variant
• CNV – copy number variant
Comparisson with reference sequence (genome)
DNA re-sequencing
CEITEC at Masaryk University 3
• DNA sequencing without selection
• Fragmentation
– Ultrasound
– Enzymes
– Longer fragments (> 300bp)
• PCR-free – GC-bias
• Paired-end sekvencing
• Germline - 30x coverage ~ 100Gb
WGS – whole genome sequencing
DNA re-sequencing
CEITEC at Masaryk University 4
• Coding sequence selection
• Fragmentation
– Ultrasound
– Enzymes
– Fragments 150-250bp
• PCR
• Paired-end sequencing
WES – whole exome sequencing
DNA re-sequencing
CEITEC at Masaryk University 5
• Coding sequence selection
• Fragmentace
– Ultrasound
– Enzymes
– Fragments 150-250bp
• PCR
• Paired-end sekvenace
• Germline - >50x coverage, >3Gb
WES – whole exome sequencing
DNA re-sequencing
CEITEC at Masaryk University 6
• Coding sequence selection
• Fragmentation
– Ultrasound
– Enzymes
– Fragments 150-250bp
• PCR
• Paired-end sekvenace
• Germline - >50x coverage
Gene panels
DNA re-sequencing
CEITEC at Masaryk University 7
• Coding sequence selection
• PCR enrichment
• Single primer PCR
• Inverse PCR + capture
• Capture + probe extension
• Paired-end sequencing
• Germinální - >50x coverage
Gene panels
DNA re-sequencing
CEITEC at Masaryk University 8
• Coding sequence selection
• PCR enrichment
• Single primer extension
• Inverzní PCR + capture
• Capture + probe extension
• Paired-end sekvenace
• Germinální - >50x coverage
Gene panels
DNA re-sequencing
CEITEC at Masaryk University 9
• Coding sequence selection
• PCR enrichment
• Single primer PCR
• Inverse PCR + capture
• Capture + probe extension
• Paired-end sekvenace
• Germinální - >50x coverage
Gene panels
DNA re-sequencing
CEITEC at Masaryk University 10
• Coding sequence selection
• PCR enrichment
• Single primer PCR
• Inverse PCR
• Capture + probe extension
• Paired-end sekvenace
• Germinální - >50x coverage
Gene panels
DNA re-sequencing
CEITEC at Masaryk University 11
• Tumor x Normal
– WGS, WES, panels
– Tumor only variants
●
Tumor only
– Panels, genes
– Variants germline i somatic
– Interpretation according to VAF, anotations, ...
• UMI, Duplex-seq adaptors
– Identification of PCR duplicates
– Error correction
Somatic variants
Thank you
for your attention
www.ceitec.eu
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