0.25
0.5
0.75
1.25
1.5
1.75
2
Application of Next Generation Sequencing technology to gene expression studies. Are we there yet?
Published on Oct 11, 20118224 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
Related categories
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