Application of Next Generation Sequencing technology to gene expression studies. Are we there yet?

Published on Feb 4, 20258236 Views

István Nagy

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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Presentation

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™ 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

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

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