Boris Tichý

RNA-seq

RNA-seq
Genes/exons sequence and expression
• “Clasical” RNA-seq
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Whole transcriptome

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PolyA selection

rRNA depletion
3’ RNA-seq
targeted RNA sequencing
single-cell RNA sequencing
smallRNA sequencing
long-read RNA sequencing
direct RNA sequencing
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CEITEC at Masaryk University

2

RNA-seq
„classical“ RNA-seq
• RNA sequencing w/wo selection
• Fragmentation
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temperature/chemic

primers
• Reverse transcription – random primers
• Double-stranded cDNA
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directional/non-directional
• Adaptor ligation
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CEITEC at Masaryk University

3

RNA-seq
„classical“ RNA-seq
• RNA selection
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polyA (mRNA)
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beads
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primer

rRNA depletion
Differential expression
Splicing analysis
De-novo transcriptome
Variant analysis
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CEITEC at Masaryk University

4

RNA-seq
3‘ RNA-seq
• 1 mRNA = 1 sequence
vs. 1 mRNA = n sequences
• Differential expression
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CEITEC at Masaryk University

5

RNA-seq
Targeted RNA-seq
• Selection of genes/exons
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During RT

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PCR/SPE after RT

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Capture after library prep

CEITEC at Masaryk University

6

RNA-seq
Single-cell RNA-seq
• 3’ sequencing / whole transcript
• Droplets, microfluidics, nanowells, plates
• UMI, cell barcodes
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CEITEC at Masaryk University

7

RNA-seq
SmallRNA sequencing
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RNA adaptors ligation
CATS
Size selection
miRNA, piRNA, ...

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CEITEC at Masaryk University

8

RNA-seq
Long-read + direct RNA sequencing
• PacBio
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Iso-seq

Isoform analysis
• Oxford Nanopore
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Direct RNA sequencing

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Isoform analysis

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Base modifications

CEITEC at Masaryk University

9

16S, RAD

Metagenomics
Analýza komplexních populací
• Shotgun metagenome
• Markers
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16S rDNA

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18S, ITS, COX

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Short vs. long amplicons

CEITEC at Masaryk University

11

RAD, ddRAD
Restriction site associated DNA markers
• Analýza variability populací
• ddRAD = double digest
• Alternativa – capture enrichment
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CEITEC at Masaryk University

12

CEITEC
@CEITEC_Brno

Thank you
for your attention
www.ceitec.eu