Genomics: Experimental Methods: Dr. Pragasam Viswanathan Professor, SBST
Genomics: Experimental Methods: Dr. Pragasam Viswanathan Professor, SBST
Genomics: Experimental Methods: Dr. Pragasam Viswanathan Professor, SBST
• Roche (454)
–Emulsion PCR
–Polymerase
–Natural Nucleotides
• 100-500 Mb for 5-15k
–1% error rate
–Homopolymers
One additional
insight ...
Short Read Techologies
• ABI
SOLID
Other second generation technology: (ABI) SOLID
Second generation DNA/RNA profiling
Second Generation DNA profiling
• Enrichment Sequencing
• ChIP-Seq (Chromosome
Immunoprecipitation)
• A substitute for ChIP-chip
• Eg. to find the binding sequence of proteins
(TFBS)
Second Generation DNA profiling
Second Generation DNA profiling
UGAGGUAGUAGGUUGUAUAGU
C.elegans let-27; 21 nt
(Pasquinelli et al. Nature 408:86-89,2000)
Often small
Sometimes multicopy and redundant Often
not polyadenylated
(not represented in ESTs)
Immune to frameshift and nonsense
mutations
No open reading frame, no codon bias
Often evolving rapidly in primary sequence
Second Generation RNA profiling
• Comparing to microarray
– Microarray
• Closed technology: Prior knowledge required
• Affected by pseudo-genes (homologous of real genes)
• Low sensitivity
– RNA-Seq
• Open technology: No prior knowledge required
• Not affected by pseudo-genes because exact
sequence is measured
• Other information could be yielded (SNP,
Alternative
splicing)
Next generation sequencing
-Third generation sequencing
Ultra-low-cost SINGLE molecule sequencing
Pacific Biosciences: A Third Generation Sequencing Technology
Complete genomics
Nanopore Sequencing
Second Generation Protein profiling
• Proteomics MS-MS-based
exclusively in discovery mode
MS/MS identification
pipeline overview
pipeline
Goal
Bonanza
Bonanza + IggyPep
Second Generation Protein profiling
3 000 000
MethylCap_Seq
6 000
EpiHealth
50
Deep_Seq
• Space/time continuum …
Thank you!!!!!!!!!