1. Next-generation sequencing
Joel Puig Sadurni

2. Introduction
Whole genome sequences of tens of species and individuals.
Gene expression profiling in multiple conditions, tissues, individuals and species
Mapping of functional regions in the genome
The advent of next generation sequencing technologies has opened new possibilities in the analysis of human disease.

3. SANGER
Sanger sequencing has been the universally used sequencing technology since its publication in 1977.
One of the strengths of Sanger sequencing is the very low error rate, and is still considered the gold standard for nucleic acid sequencing. New mutations identified by NGS technologies are validated using Sanger sequencing

4. Second generation technologies
Second generation technologies, also called high-throughput or NGS technologies, have exponentially increased the quantity of sequences generated, producing up to several million bases (Gb) in a single run
the main concept behind them is similar (library preparation)

5. Third generation sequencing technologies
The race for the 1000$ genome in 1 day
The main current limitation is the higher error rate.
PacBio RS (Pacific Biosciences)
GridION (Oxford Nanopore Technologies)
Ion Torrent ( Life Technologies Corporation)

6. PacBio RS (Pacific Biosciences)
Single molecule, real-time(SMRTTM) detection
provides direct measurement of individual molecules
• Templates can be prepared for the PacBio RS
without PCR amplification, resulting in more uniform
sequence coverage across genomic regions
regardless of GC content, facilitating the detection of
minor variants in heterogeneous samples
• While observing single molecule sequencing by a
highly processive strand-displacing DNA polymerase
in real time, the system also records the kinetics of
each nucleotide incorporation reaction, identifying
base modifications of the native templates, such as
DNA methylation

7. GridION (Oxford Nanopore Technologies)

8. GridION (Oxford Nanopore Technologies)
If you can put 20 of the GridIONs together you can sequence the entire human genome in 15 minutes for about $1500 per genome.
A lot of the interest has come from the (very cool) MinION, miniaturized version of the GridION, disposable USB-key sequencer that can sequence about a billion base pairs of DNA, and cost around $500-$900 each.

9. Ion Torrent ( Life Technologies Corporation)

10. Ion Torrent ( Life Technologies Corporation)
An integrated semiconductor device enabling non-optical genome sequencing. Rothberg JM et al. Nat Methods. 2011;8(9): PMID:

11. Ion Torrent ( Life Technologies Corporation)

12. Ion Torrent ( Life Technologies Corporation)
The Personal Genome Machine (PGM™) Sequencer: €
Sequencing Scale:3 Mb to 1 Gb 
Read Length: bases (average: 100 bases) 
Run Time: 2 hrs (average)
Ion Proton™ Sequencer €
Throughput: Up to 10 Gb (Note: The Ion Proton™ II Chip will be available about six months after the Ion Proton™ I Chip. The Ion Proton™ II Chip will enable sample-to-variant analysis of a human genome in a single day, at up to 20x coverage.)
Read length:Up to 200-base fragment reads
Sequencing run time: As little as 2 hours for 100-base reads

13. Aplications Targeted Resequencing De Novo Assembly
Pathogen Identification
Transcript Analysis
Genetic human variation : SNP, CNV
Comparative studies
Epigenomics
Identification of all mutations in an organism
Chip-seq: interactions protein-DNA
Ancient DNA
DNA mixtures from diverse ecosystems, metagenomics
Resequencing previously published reference strains
Errors in published literature
Expand the number of available genomes
Sequencing extremely large genomes
Detection of cancer specific alleles avoiding traditional cloning
Detecting ncRNA

14. Bibliography
An integrated semiconductor device enabling non-optical genome sequencing. Rothberg JM et al. Nat Methods. 2011;8(9):708-9.
Next-generation sequencing technologies for environmental DNA research. Shokralla S. et al. Molecular Ecology (2012) 21,
Performance comparison of benchtop high-throughput sequencing platforms. Loman N. et al. Nature biotechnology (2012) V.30 number 6