RNA-seq
Boris Tichý
RNA-seq
CEITEC at Masaryk University 2
• “Clasical” RNA-seq
– Whole transcriptome
– PolyA selection
– rRNA depletion
• 3’ RNA-seq
• targeted RNA sequencing
• single-cell RNA sequencing
• smallRNA sequencing
• long-read RNA sequencing
• direct RNA sequencing
Stanovení sekvence a exprese genů/exonů
RNA-seq
CEITEC at Masaryk University 3
• Sekvenace RNA s/bez selekce
• Fragmentace
– teplota/chemicky
– primery
• Reverzní transkripce – random primery
• Double-stranded cDNA
– directional/non-directional
• Ligace adaptorů
„classical“ RNA-seq
RNA-seq
CEITEC at Masaryk University 4
• Selekce
– polyA (mRNA)
●
beads
●
primer
– rRNA deplece
• Differential expression
• Splicing analysis
• De-novo transcriptome
• Variant analysis
„classical“ RNA-seq
RNA-seq
CEITEC at Masaryk University 5
• 1 mRNA = 1 sekvence
– vs. 1 mRNA = n sekvencí
• Differential expression
•
3‘ RNA-seq
RNA-seq
CEITEC at Masaryk University 6
• Selekce vybraných genů
• Během Rev. transkripce
• PCR/SPE po cDNA syntéze
• Capture po přípravě knihoven
•
Targeted RNA-seq
RNA-seq
CEITEC at Masaryk University 7
• 3’ sequencing / whole transcript
• Droplets, microfluidics, nanowells, plates
• UMI, cell barcodes
•
•
•
Single-cell RNA-seq
RNA-seq
CEITEC at Masaryk University 8
• Ligace RNA adaptorů
• CATS
• Size selection
• miRNA, piRNA, ...
•
SmallRNA sequencing
RNA-seq
CEITEC at Masaryk University 9
• PacBio
– Iso-seq
– Isoform analysis
• Oxford Nanopore
– Direct RNA sequencing
– Isoform analysis
– Base modifications
Long-read + direct RNA sequencing
16S, RAD
Metagenomics
CEITEC at Masaryk University 11
• Shotgun metagenome
• Markers
– 16S rDNA
– 18S, ITS, COX
– Short vs. long amplicons
Analýza komplexních populací
RAD, ddRAD
CEITEC at Masaryk University 12
• Analýza variability populací
• ddRAD = double digest
• Alternativa – capture enrichment
•
Restriction site associated DNA markers
Thank you
for your attention
www.ceitec.eu
CEITEC
@CEITEC_Brno