1. Introduction to NGS

4. The Human Genome Draft: Cutting in Pieces and Rejoining in Sequencesb
Fragment, insert (2-3 Kb) into BAC clones, amplify, extract (8-fold oversampling, to reach coverage)
Excide, insert (200 b) into plasmids, amplify, extract
 Sanger sequence (bottleneck)

5. DNA sequencing: the bottleneck

7. The 3 phases of NGS

9. REPAIR END
-Purification (- little fragm. TE)
-T4 DNA POL./ KLENOW
-PNK (5’)
-MIX BUFFER AND ENZYMES
- T= 30 min; 20°C (DON’T CLOSE LID)
- PURIFICATION (ETOH!)

10. ADENILATION
- KLENOW EXO-
- MIX BUFFER AND ENZYMES
- T= 30 min; 37°C (DON’T CLOSE LID)
- PURIFICATION (IMMEDIATELY) MINI
ELUTE (Q)- room temperature

11. LIGATE ADAPTER
- DNA LIGASE
- T= 15 min; 20°C (DON’T CLOSE LID)
- PURIFICATION PCR PUR. KIT- room
temperature
-NTC?

12. SELECTION SIZE RANGE
- AGAROSE 2% TAE
- i.e. 400 bp +/- 5% ( bp): 2 mm
- i.e. Read 2x75 bp: insert 200 bp; to cut 300 bp
To avoid overlapping
- i.e. Read 2x100 bp: insert 300 bp; to cut 400 bp
- PURIFICATION (gel extraction) (no step 50°C)

13. ENRICH DNA FRAGMENTS
-PCR REACTION (10 CYCLES)
-Primer P1 and P2 anneal to the ends of adaptors
-Additional sequences are added enable
hybridization with primers bound to the flow cell
-to enrich DNA fragments with adapters on both
ends
- to enable reliable quantitation
- PURIFICATION

14. LIBRARY VALIDATION
- OPTICAL DENSITY/ INTERCALATING DYE
- BIOANALYZER
- qPCR

17. WHOLE GENOME RESEQUENCING
to sequence the individuals when the whole genome sequence of that speciesis already known.
TARGETED RESEQUENCING
- to sequence specific regions of the individuals when the whole genome
sequence of that species is already known.
CHROMATIN IMMUNOPRECIPITATION SEQUENCING (ChIP-Seq)
ChIP-Seq is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest.
DE NOVO SEQUENCING
- the initial generation of the primary genetic sequence
of a particular organism

22. RNA-seq Transcriptome profiling based on deep-sequencing technologies:
–RNA-seq provide a «digital» measure of transcripts levels
–Detection of alternative splicings
–Detection of polymorphisms in expressed regions
–Does not need an a priori knowledge of gene positions (transcripts reconstruction, detection of unannotated transcripts…)
–Higher dinamic range compared to microarrays

28. Nature Methods 6, 711 - 713 (2009) doi:10.1038/nmeth1009-711
Is sequencing enlightenment ending the dark age of the transcriptome?
Piero Carninci1
Abstract
Sequencing-based technologies for RNA discovery are playing a key role in deciphering the transcriptome and hold the potential to provide us with a census of RNAs and their functions.
RNA Biol. 2009 Apr-Jun;6(2):
Whole genome transcriptome analysis.
Forrest AR, Carninci P.
RIKEN Omics Science Center, RIKEN Yokohama Institute, Kanagawa, Japan.
Abstract
Recent studies indicate that the majority of the genome is transcribed, however only a fraction of this transcription is annotated as what we traditionally know as genes. Much of this is low level pervasive, and often overlapping transcription, for which the transcript structure and function, has not as yet been determined. Novel RNAs (and novel classes of RNAs), are now being identified by groups using next generation sequencing and cDNA libraries that target specific sub-fractions of the transcriptome (based upon size, modifications, localization and protein interactions). We discuss the state of the art as to measuring and identifying these novel RNAs and speculate on whether a universal approach to transcript isoform and expression level measurement is possible in the near future.

31. Disadvantages
Price
Mistakes of Polimerase Preamplification
Data Analysis Mistakes
Validations data

33. Data Analysis
96 CPU
2Tbite Ram
200Tbite Hard disk

34. SMRT Sequencing Advantages
Sequencing with the PacBio RS system based on our SMRT technology offers the following key benefits:
• Single molecule, real-time analysis. SMRT technology harnesses the power of the DNA polymerase to enable single molecule, real-time sequencing. The ability to resolve single molecules in real time allows our system to observe structural and cell type variation not accessible with other technologies. Unlike existing sequencing platforms, minimal amounts of reagent and sample preparation are required and there are no time-consuming flushing, scanning and washing steps. In addition, our platform does not require the routine PCR amplification needed by most second generation sequencing systems thereby avoiding systematic amplification bias.

35. SPECIAL OFFER
€ 12,500 for human whole genome sequencing minimum 80 samples
€ 3,900 for human exome sequencing minimum 10 samples
€ 1,100 for whole human transcriptome sequencing minimum 10 samples
€ 950 for human miRNA sequencing minimum 10 samples

36. I happy I'm feeling glad I got sunshine in a bag I'm useless but not for long the future is coming on