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Application of Next Generation Sequencing technology to gene expression studies. Are we there yet?
Published on 2011-10-118224 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
Presentation
Next-generation Sequencing00:00
Sanger sequencing07:09:20
Comparison of technologies35:54:40
2 Base Encoding70:23:27
Instrument style81:01:42
Continued Innovation of the SOLiD™ System105:06:26
Life Technologies™ Sequencing Portfolio117:26:34
The thousand dollar genome136:54:33
The Race is On163:18:33
SOLiD™ 5500/xl Workflow168:02:32
SOLiD™ Workflow201:33:11
Assembling complex genomes: 2 problems259:55:12
Assembling problems in complex genomes272:36:50
Available Library Protocols Three Methods Available for Different Applications289:09:30
SOLiD™ Mate-Paired Library Workflow372:32:02
SOLiD™ Mate-Paired Library Workflow383:02:38
SOLiD™ Chemistry (1)402:42:18
SOLiD™ Chemistry (2)432:26:19
ePCR: Oil Phase Premix automatization434:05:23
Sequencing Reagents in New, Easy-to-Use Format (1)439:50:33
Sequencing Reagents in New, Easy-to-Use Format (2)453:10:05
Substrate Preparation (1)479:18:37
Substrate Preparation (2) 483:14:32
Substrate Preparation (3)484:01:18
SOLiD™ Chemistry485:58:33
Properties of the Probes516:16:05
SOLiD 4-color ligation Ligation reaction (1)537:49:22
SOLiD 4-color ligation Ligation reaction (2)544:48:14
SOLiD 4-color ligation Visualization545:40:39
SOLiD 4-color ligation Cleavage556:27:15
SOLiD 4-color ligation Ligation (2nd cycle) 558:31:40
SOLiD 4-color ligation Visualization (2nd cycle) 561:20:52
SOLiD 4-color ligation Cleavage (2nd cycle)566:42:06
SOLiD 4-color ligation573:16:02
SOLiD 4-color ligation Reset575:24:19
SOLiD 4-color ligation (1st cycle after reset) - 1579:24:45
SOLiD 4-color ligation (1st cycle after reset) - 2584:39:21
SOLiD 4-color ligation (2nd Round)593:01:50
Sequential rounds of sequencing Multiple cycles per round593:27:10
2 base pair encoding reference alignment in color space619:48:36
Advantages of 2 base pair encoding644:19:34
2 base pair encoding reference alignment in color space647:01:28
Advantages of 2 base pair encoding Miscall656:07:31
But there is more… only certain transitions are allowed for a real SNP661:58:31
The only allowed transitions665:31:10
The following color changes are not allowed, as 2 bases change667:35:00
Flexibility674:27:53
Multiplexing Enables to Run Up to 256 Samples / Run Scalability for (small) RNA Expression, Targeted Re-Sequencing, etc.676:58:21
SOLiD™ System Barcodes683:56:17
Multiplex Analysis696:51:05
Workflow697:20:12
The SOLID Workflow697:53:43
SOLiD Resequencing Workflow (1)705:36:59
SOLiD Resequencing Workflow (2)705:48:39
Workflow analysis (WFA)708:34:27
Types of sequencing runs733:50:15
Experimental Design747:30:12
SOLiD Applications774:08:47
Genome Sequencing (1)780:20:07
Genome Sequencing (2)785:12:30
NGS Genome Sequencing Shotgun Startegy789:05:34
Resequencing: Genome Comparison803:52:30
De Novo Sequencing (reference unavailable)819:55:57
CLC GW de novo assembly; hybrid datasets820:16:23
CLC Genomics Workbench de novo assembly zoom out (1)835:51:18
CLC Genomics Workbench de novo assembly zoom out (2)840:15:02
Metagenomics850:24:40
The Complexity of the Transcriptional Landscape871:38:17
Microarray vs. SAGE vs. TaqMan vs. NGS879:46:21
Genes from the reference (retain annotations)903:21:59
Outcome (1)920:42:49
Outcome (2)929:59:08
Outcome (3)933:24:31
Outcome (4)939:18:26
SOLiD Whole Transcriptome Kit962:27:15
Example of “strandedness”973:12:23
Epigenetic Complexes979:02:02
Fragment workflow for ChIP-Seq using SOLiD™ System986:19:26
ChIP-Seq Results993:59:52
ChIP comparison; experimental replicates999:12:56
ChIP comparison; background999:31:55
Targeted resequencing999:33:20
Create a Bait Group1009:57:01
SureSelectTarget Enrichment System Workflow1010:32:00
SETS: Satay report1029:53:10
SETS: N2S report1030:16:26
Resequencing1030:57:12
"All animals are equal, but some animals are more equal than others!"1053:39:40
Inflammatory acne1056:47:32
Pathogenic factors in acne1060:11:28
Innate immunity in the skin1072:07:13
Distinct P.acnes strains induce selective gene expression in keratinocytes1086:58:51
Comparison of P. acnes isolates by fermentation and immunofluorescene1102:03:38
Unrooted phylogenic tree of P. acnes isolates based on the recA gene sequence1103:32:58
RAPD – random amplification of polymorphic DNA (1)1111:24:50
RAPD – random amplification of polymorphic DNA (2)1131:48:06
~48kb deletion spanning 35 genes confirmed by capseq (1)1143:13:52
~48kb deletion spanning 35 genes confirmed by capseq (2)1152:32:43
Resistance to antibiotics1163:33:45
Variability in codon usage (1)1209:25:53
Variability in codon usage (2)1216:32:36
Digital Gene Expression (DGE) analysis of PGN treated dendritic cells1216:51:36
SOLiD DGE workflow1220:28:24
Experimental layout1257:42:32
GO-term enrichment (iDC: Unstimulated - Stimulated)1281:26:52
GO-term enrichment (Immature - Mature)1295:21:48
Validation (1)1295:53:09
Validation (2)1309:57:59
Validation (3)1318:23:21
TNF-α1328:53:54
TNF family members1334:31:49
Pathway example (Unstimulated vs. ONE PGN Stimulation)1354:49:18
Pathway example (Unstimulated vs. TWO PGN Stimulations)1368:30:43
Pathway example (Immature vs. Mature) - 11375:06:51
Pathway example (Immature vs. Mature) - 21377:25:12
Whole transcriptome analysis (RNA-Seq) of neutrofil-like cells treated with Chlamydia trachomatis1379:17:18
The Complexity of the Transcriptional Landscape1386:52:46
SOLiD Whole Transcriptome Analysis workflow1387:12:35
Experimental layout (1)1401:45:15
Experimental layout (2)1422:05:41
Cytokines (1)1423:24:19
Cytokines (2)1437:30:28
Members of the proteasome complex1443:37:31
Antigen presentation (1)1456:35:24
Antigen presentation (2)1470:35:46
Next Generation Applications1485:50:21
Partners1502:22:34
Lab1523:36:55
Thank you for your attention!1531:27:19