Curso Dr. Alvarez

1. Next Generation Sequencing

7. •One Bead
•One DNA library
•NTPs, Taq etc.

15. 3 Main Technologies
Solid

25. http://www.dkfz.de/gpcf/849.html

29. http://www.flickr.com/photos/doe_jgi/4093644608

31. The development and impact of 454 sequencing Jonathan M Rothberg & John H Leamon Nature Biotechnology 26, 1117 - 1124 (2008) Published online: 9 October 2008
doi:10.1038/nbt1485

38. Ion Torrent - PGM/Proton
• The Ion Torrent System
– Two instruments: PGM and Proton
– For small scale (PGM) and large scale sequencing (Proton)
– Rapid sequencing (run time ~ 2-4 hours)
– Measures changes in pH
– Sequencing on a chip
Personal Genome Machine (PGM) Ion Proton

47. Ion Torrent output
• Ion Torrent throughput
~ from 10Mb to >10Gb, depending on the chip
• Read lengths: 400bp (PGM), 200bp (Proton)
• Output file format: FASTQ
• What can we use Ion Torrent for?
– Anything, except perhaps very large genomes
2 human exomes (PI chip)
2 human transcriptomes
1 human genome = 6 PI chips
PI
(Proton)
318
(PGM)
316
(PGM)
314
(PGM)
NEW! P2
(Proton)

48. Ion Torrent analysis workflow
Downstream analysis
Torrent Server
.fastq
.bam
.fasta

49. So…What’s next?

51. Overview
• Similar idea as SMRT
• Detects the
chemiluminescence given off
when a proton (H+) is released
•Use ion chip

52. Library Construction
If RNA, converted into cDNA
DNA fragmentation
Adapter ligated to DNA
Denaturation
Image provided by http://www.thermoscientificbio.com/ngs-library-preparation-kits/

53. Template Prep/ePCR Amplification
Fragments are attached to beads
ePCR

54. Sequencing
Based on standard pyrosequencing
Measures light from chemiliuminescent reagents
by measuring direct release of H+ (protons) from
the sequencing reaction

55. Data Analysis
Output  FASTQ
Image provided by http://mendel.iontorrent.com/ion-docs/On-Dataflow.html