Recommended
More Related Content
What's hot
What's hot (20)
Similar to Introduction to NGS
Similar to Introduction to NGS (20)
More from cursoNGS
More from cursoNGS (9)
Recently uploaded
Recently uploaded (20)
Introduction to NGS
- 1. Introduction to NGS Ana Conesa Head of Genomics of Gene Expression Lab Centro de Investigaciones Prínicpe Felipe aconesa@cipf.es http://bioinfo.cipf.es/aconesa
- 2. Next Generation Sequencing NGS has brought high speed not only to genome sequencing and personal medicine, but has also change the way we do genome research: Got a question on genome organization: SEQUENCE IT!!!!
- 3. NGS technologies Cost-effective Fast Ultra throughput Cloning-free Short reads
- 6. Roche 454
- 8. Solexa
- 9. Solexa
- 10. Solexa-HiSeq 200 Gb/run in 8 days 2x100 bp fragments 2 billion reads per run
- 11. Helicos
- 12. SOLiD
- 13. SOLiD * Sequencing output in “color space” * Needs reference genome to translate to base space.
- 14. SOLiD 5500 * Fifth 3-based encoded primer * Sequencing output in base space * No reference needed
- 15. 5500 xl-u SOLiD 180 Gb/run (microbeads) 300 Gb/run (nanobeads) 35-75 bp fragments 2.8 - 4.8 billion reads/run 2x6 lanes/run 96 bar-codes 99.99% accuracy
- 16. Pacific Biosystems Real time DNA synthesis Up to 12000 nt?? 50 bases/second??
- 17. Ion Torrent •$ 50.000 •$ 500 /sample • 1 hour/run • > 200 nt lengths •Reads H+ released by DNA polymerase
- 18. Comparison Roche 454 Solexa SOLiD •Long fragments •Short fragments •Short fragments •Errors: poly nts •Errors: Hexamer bias •Color-space •Low throughput •High throughput •High throughput •Expensive •Cheap •Cheap •De novo sequencing: •Resequencing: •Resequencing: •Amplicon sequencing •ChipSeq •ChipSeq •RNASeq •RNASeq •MethylSeq •MethylSeq
- 19. Applications De novo sequencing Resequencing Exome Sequencing RNA-seq Genome annotation Chip-seq Methyl-seq …….
- 20. Applications De novo sequencing Resequencing Exome Sequencing RNA-seq Genome annotation Chip-seq Methyl-seq …….
- 21. Basic steps NGS data processing QC and read cleaning
- 22. Basic steps NGS data processing QC and Mapping read cleaning
- 23. Basic steps NGS data processing QC and Feature Mapping read cleaning identification
- 24. Basic steps NGS data processing SNVs Indels Rearrang. QC and Feature RPKM Mapping Splicing read cleaning identification DNA Binding site
- 25. RNA-seq Elucidate gene models Quantify gene expression
- 26. RNA-seq Elucidate gene models
- 27. RNA-seq protocol* total RNA purification mRNA preparation oligodT RiboZ 2nd strand synthesis 1st strand synthesis fragmentation RNA DN *Solexa Pair-End A
- 28. RNA-seq protocol (II) 100bp lad A A adenylation 3’ ends A A A A A A A A 400-200 ligate adapters amplification library 400-200 SEQUENCING!
- 31. File formats fastq: sequence data and qualities SAM/BAM: mapping data and qualities
- 32. Some Figures How much does it “cost” (computationally) to sequence a human transcriptome? One human transcriptome: 100 Million reads 1 Solexa run ==8 lanes ==25 M reads/lane==2 x 4 G fastq/lane (PE) 32 G disk space Mapping @ processor 12 cores, 48 GB RAM , 4TB disk 24 hours SAM (Ascii) / BAM (Binary) output 36 G / 9 G
- 33. Applications of RNAseq Qualitative: Quantitative: * Alternative splicing * Differential expression * Antisense expression * Dynamic range of gene expression * Extragenic expression …. * Alternative 5’ and 3’ usage * Detection of fusion transcripts …. edgeR Tophat/Cufflinks DESeq Scripture baySeq Alexa NOISeq
- 34. Advantages of RNAseq? RNAseq microarrays * Non targeted transcript detection * Restricted to probes on array * No need of reference genome * Needs genome knowledge * Strand specificity * Normally, not strand specific * Find novels splicing sites * Exon arrays difficult to use * Larger dynamic range * Smaller dynamic range * Detects expression and SNVs * Does not provide sequence info * Detects rare transcripts * Rare transcripts difficult …. …. and…. are there any disadvantages?????
- 35. Resequencing
- 36. Exome Sequencing Gene A Gene B DNA (patient) 1 Produce shotgun library 2 Determine Capture exon sequences Map against 5 variants, 4 reference Filter, compare genome 3 patients candidate genes Wash & Sequence
- 37. Exome capture
- 38. The principle: comparison of patients Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 candidate gene (shares mutation for all patients) mutation
- 39. ChipSeq
- 40. MethylSeq
- 41. MIDseq
- 46. Miller syndrome
- 49. Species composition of metagenomic DNA extracted from mammoth hair.
- 50. Conclusions NGS is revolutionizing how we do genome research
- 51. Conclusions NGS is revolutionizing how we do genome research But it will also revolutionize our lives….
- 52. Conclusions NGS is revolutionizing how we do genome research But it will also revolutionize our lives…. If we manage to process and analyze the data
- 53. YOUR SUCESSFUL STORY??? Have a great MDA course?