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Application of Next Generation Sequencing technology to gene expression studies. Are we there yet?
Published on Oct 11, 20118231 Views
Novel high-throughput deep sequencing technology has dramatically changed the way that the functional complexity of genomes and transcriptomes can be studied. In addition, SOLiD platform provides two
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Chapter list
Next-generation Sequencing00:00
Sanger sequencing00:25
Comparison of technologies02:09
2 Base Encoding04:13
Instrument style04:51
Continued Innovation of the SOLiD™ System06:18
Life Technologies™ Sequencing Portfolio07:02
The thousand dollar genome08:12
The Race is On09:47
SOLiD™ 5500/xl Workflow10:04
SOLiD™ Workflow12:05
Assembling complex genomes: 2 problems15:35
Assembling problems in complex genomes16:21
Available Library Protocols Three Methods Available for Different Applications17:20
SOLiD™ Mate-Paired Library Workflow22:21
SOLiD™ Mate-Paired Library Workflow22:58
SOLiD™ Chemistry (1)24:09
SOLiD™ Chemistry (2)25:56
ePCR: Oil Phase Premix automatization26:02
Sequencing Reagents in New, Easy-to-Use Format (1)26:23
Sequencing Reagents in New, Easy-to-Use Format (2)27:11
Substrate Preparation (1)28:45
Substrate Preparation (2) 28:59
Substrate Preparation (3)29:02
SOLiD™ Chemistry29:09
Properties of the Probes30:58
SOLiD 4-color ligation Ligation reaction (1)32:16
SOLiD 4-color ligation Ligation reaction (2)32:41
SOLiD 4-color ligation Visualization32:44
SOLiD 4-color ligation Cleavage33:23
SOLiD 4-color ligation Ligation (2nd cycle) 33:30
SOLiD 4-color ligation Visualization (2nd cycle) 33:40
SOLiD 4-color ligation Cleavage (2nd cycle)34:00
SOLiD 4-color ligation34:23
SOLiD 4-color ligation Reset34:31
SOLiD 4-color ligation (1st cycle after reset) - 134:45
SOLiD 4-color ligation (1st cycle after reset) - 235:04
SOLiD 4-color ligation (2nd Round)35:34
Sequential rounds of sequencing Multiple cycles per round35:36
2 base pair encoding reference alignment in color space37:11
Advantages of 2 base pair encoding38:39
2 base pair encoding reference alignment in color space38:49
Advantages of 2 base pair encoding Miscall39:22
But there is more… only certain transitions are allowed for a real SNP39:43
The only allowed transitions39:55
The following color changes are not allowed, as 2 bases change40:03
Flexibility40:28
Multiplexing Enables to Run Up to 256 Samples / Run Scalability for (small) RNA Expression, Targeted Re-Sequencing, etc.40:37
SOLiD™ System Barcodes41:02
Multiplex Analysis41:48
Workflow41:50
The SOLID Workflow41:52
SOLiD Resequencing Workflow (1)42:20
SOLiD Resequencing Workflow (2)42:20
Workflow analysis (WFA)42:30
Types of sequencing runs44:01
Experimental Design44:51
SOLiD Applications46:26
Genome Sequencing (1)46:49
Genome Sequencing (2)47:06
NGS Genome Sequencing Shotgun Startegy47:20
Resequencing: Genome Comparison48:13
De Novo Sequencing (reference unavailable)49:11
CLC GW de novo assembly; hybrid datasets49:12
CLC Genomics Workbench de novo assembly zoom out (1)50:09
CLC Genomics Workbench de novo assembly zoom out (2)50:24
Metagenomics51:01
The Complexity of the Transcriptional Landscape52:17
Microarray vs. SAGE vs. TaqMan vs. NGS52:47
Genes from the reference (retain annotations)54:12
Outcome (1)55:14
Outcome (2)55:47
Outcome (3)56:00
Outcome (4)56:21
SOLiD Whole Transcriptome Kit57:44
Example of “strandedness”58:23
Epigenetic Complexes58:44
Fragment workflow for ChIP-Seq using SOLiD™ System59:10
ChIP-Seq Results59:38
ChIP comparison; experimental replicates59:57
ChIP comparison; background59:58
Targeted resequencing59:58
Create a Bait Group01:00:35
SureSelectTarget Enrichment System Workflow01:00:37
SETS: Satay report01:01:47
SETS: N2S report01:01:48
Resequencing01:01:51
"All animals are equal, but some animals are more equal than others!"01:03:13
Inflammatory acne01:03:24
Pathogenic factors in acne01:03:36
Innate immunity in the skin01:04:19
Distinct P.acnes strains induce selective gene expression in keratinocytes01:05:13
Comparison of P. acnes isolates by fermentation and immunofluorescene01:06:07
Unrooted phylogenic tree of P. acnes isolates based on the recA gene sequence01:06:12
RAPD – random amplification of polymorphic DNA (1)01:06:41
RAPD – random amplification of polymorphic DNA (2)01:07:54
~48kb deletion spanning 35 genes confirmed by capseq (1)01:08:35
~48kb deletion spanning 35 genes confirmed by capseq (2)01:09:09
Resistance to antibiotics01:09:48
Variability in codon usage (1)01:12:33
Variability in codon usage (2)01:12:59
Digital Gene Expression (DGE) analysis of PGN treated dendritic cells01:13:00
SOLiD DGE workflow01:13:13
Experimental layout01:15:27
GO-term enrichment (iDC: Unstimulated - Stimulated)01:16:53
GO-term enrichment (Immature - Mature)01:17:43
Validation (1)01:17:45
Validation (2)01:18:35
Validation (3)01:19:06
TNF-α01:19:44
TNF family members01:20:04
Pathway example (Unstimulated vs. ONE PGN Stimulation)01:21:17
Pathway example (Unstimulated vs. TWO PGN Stimulations)01:22:06
Pathway example (Immature vs. Mature) - 101:22:30
Pathway example (Immature vs. Mature) - 201:22:38
Whole transcriptome analysis (RNA-Seq) of neutrofil-like cells treated with Chlamydia trachomatis01:22:45
The Complexity of the Transcriptional Landscape01:23:12
SOLiD Whole Transcriptome Analysis workflow01:23:13
Experimental layout (1)01:24:06
Experimental layout (2)01:25:19
Cytokines (1)01:25:24
Cytokines (2)01:26:15
Members of the proteasome complex01:26:37
Antigen presentation (1)01:27:23
Antigen presentation (2)01:28:14
Next Generation Applications01:29:09
Partners01:30:08
Lab01:31:25
Thank you for your attention!01:31:53