Aplicaciones y limitaciones de las tecnologías Next Generation

Sequencing a nuestros estudios

Sanger sequencing

Sanger sequencing

Number of sequences 96 Sequences/runLength 0,5-2 KiloBases

Accuracy 99%Cost 200.000 $ GigaBase

Sanger sequencing

Sanger sequencing

• Published in 2003.• Free access.• 3 Billons $ and 15 years of work.• Starting point for future projects (Encode,

1000 Genomes…).• Economic impact until 2011: 796 Billons $.

Sanger sequencing

• However:1. The cost of a new genome still high,

10.000 – 100.000 $.2. Hard lab work.3. Development of new technologies are

essentials.

Next Sequencing Technologies

Next Sequencing Technologies

Next Sequencing Technologies

Sanger NGSNumber of sequences

96 Seqs/run 8 Human Genomes/run

Length 0.5-2 KiloBases Up to 40 KiloBasesAccuracy 99% 85% to 99%

Cost 200.000 $ GigaBase

2.000 $ GigaBase

Next Next Sequencing Technologies

New challenges

• Bioinformatic approach, how work with Gbytes of sequences?? Linux

New challenges

• Bioinformatic approach, how work with Gbytes of sequences?? Linux

• Quality controls.• Decision in sequencing platform and software. • Constant change. First NGS technologies (e.g.

Roche 454) are obsolete.

New challenges

• Number of samples.• Necessity of replicates? Biological –

Technical?• Type of reads.• Number of reads / Coverage.• Library construction and complexity.• Reference genome. Gene annotation.

Some applications: Assembly de novo

Some applications: Metagenomics

• Characterize species (bacteria/virus) present in an environment• Soil, water, fecal…

• Associate metagenomics results with the origin of the sample (e.g. host, environment etc.).

• Sequence of specific region (e.g. 16S), not whole genome.

• Binning with described species.• Specific software: Phymm, MetaPhlAn…

Some applications: Metagenomics

• Binning with described species.

Some applications: Metagenomics

• Increase bacterial genome sequences.• 4.100 species. 17.000 bacterial genomes.

Some applications: Population genomics

• Whole genome not necessary for all cases.• Identify neutral and adaptive regions.• Not necessary reference genome.• Different approaches, based in restriction

enzymes:• Genotyping By Sequencing (GBS).• Restriction site Associated DNA (RAD).

Some applications: Population genomics

Some applications: Population genomics

Some applications: Transcriptomics

• Real expression of the DNA.• Not necessary reference genome.• Annotation with described genes.• Quantify genome expression.

Some applications: Transcriptomics

• Read mapping (alignment): place the “shorts” reads in the genome

• Quantification:• Assigning to genes• Determining whether a gene is expressed• Normalization• Compare between samples

Some applications: Transcriptomics

Some applications: Transcriptomics

Some applications: Transcriptomics

Some applications: Transcriptomics

Gracias…