Next-generation
sequencing
Joel Puig Sadurni
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.
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
Second generation technologies
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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)
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)
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
GridION (Oxford Nanopore
Technologies)
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.
Ion Torrent ( Life Technologies
Corporation)
Ion Torrent ( Life Technologies
Corporation)
An integrated semiconductor device enabling non-optical genome sequencing. Rothberg JM et al. Nat Methods. 2011;8(9):708-9.
PMID: 21985001
Ion Torrent ( Life Technologies
Corporation)
Ion Torrent ( Life Technologies
Corporation)
• The Personal Genome Machine (PGM™) Sequencer:
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55.000 €
Sequencing Scale:3 Mb to 1 Gb
Read Length: 35 - 200 bases (average: 100 bases)
Run Time: 2 hrs (average)
• Ion Proton™ Sequencer
– 117.000 €
– 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
Aplications
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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
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
• http://www.pacificbiosciences.com
• http://www.iontorrent.com/
• http://www.nanoporetech.com