Genomic approaches: Next Generation Sequencing by Sin Ruow Tey and Becky Reese Background image from http://0.static.wix.com/media/a69ad6_119564a450b93fdf0ea36d296d1fc79e.jpg_1024
Genomic sequencing Image by Sin Ruow Tey
First generation sequencing: Sanger Source: http://www.cytologystuff.com/images/dna2.gif
Source: http://smcg.ccg.unam.mx/enp-unam/03-estructuradelgenoma/animaciones/secuencia.swf
Sanger sequencing animation http://www.youtube.com/watch?v=oypllbi0qf8sanger
Limitations of Sanger sequencing Accuracy: http://www.socialmediaexplorer.com/wp-content/uploads/2012/04/300px-high_accuracy_low_precision.svg_.png Money: http://2.bp.blogspot.com/-itybuiqrmfs/t4g8ki14fri/aaaaaaaaaom/dyxi9xxqfae/s1600/bags_money.png Time: http://www.veryicon.com/icon/png/system/nx10/time.png
Next Generation Sequencing more accurate cheaper faster Accuracy: https://cdn1.iconfinder.com/data/icons/targetdart/506/target-dart.png Money: http://24.media.tumblr.com/232ac8b936ac9c8af59fbd464d36bdc4/tumblr_meruob5pqz1qishbfo1_500.png Hourglass: http://png-3.findicons.com/files/icons/766/base_software/256/hourglass.png
Illumina/Solexa STEP 1: Template preparation Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf
Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf Illumina/Solexa STEP 2: Attachment
Illumina/Solexa STEP 3: Bridge formation Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf
Illumina/Solexa STEP 4: Cluster generation Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf
Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf Illumina/Solexa STEP 5: Sequencing
Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf Illumina/Solexa STEP 6: Imaging
Source: http://pellegrini.mcdb.ucla.edu/lab/publication_pdfs/week1_sequencing.pdf Illumina/Solexa STEP 7: Alignment
Illumina/Solexa technology animation http://www.youtube.com/watch?v=womkfikwlxme 10
Source: http://www.nature.com/nrg/journal/v11/n1/images/nrg2626-f1.jpg Other NGS platform: Roche 454 Template preparation: emulsion PCR
Other NGS platform: Roche 454 Sequencing: pyrosequencing (Roche/454) http://www.youtube.com/watch?v=nffgwgfe0aa
Other NGS platform: Roche 454 Mapping: de novo assembly Source: http://gcat.davidson.edu/phast/img/coverage.png
Limitations of Illumina/Solexa technology need a reference genome need high coverage to detect SNPs error rate increases past 32bp Hand sign: http://3.bp.blogspot.com/_rkg-zitsnuq/tlioo9_hjfi/aaaaaaaabso/oidteyuqzac/s1600/blogger%2blimits.png
Obesity Often attributed to poor diet Can also be influenced by genetics h"p://cris*naffalcao.wordpress.com/category/obesity/
The microbiome Collec*on of microbes that co- inhabit an organism s body Human Microbiome Project (HMP) Gut microbiome poten*ally linked to obesity h"p://www.broadins*tute.org/news/4199
Obese microbiome in mice h"p://willpowerisforfatpeople.com/wp- content/uploads/2011/04/fat_mouse.jpg h"p://www.medclient.com/wp- content/uploads/2011/09/fat- mouse- skinny- mouse- obesity- longevity.jpg Background image: h"p://www.techvert.com/wp- content/uploads/2010/08/30/oil- ea*ng- microbes- consume- oil- plume/microbes.jpg Paper Referenced: Turnbaugh, P.J et al. An obesity- associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027-1031 (2006)
Le Chatelier et al. Nature 500, 541-546 (2013) doi:10.1038/nature12506
Studying the obesity/microbiome connection in humans 123 169 Dung: h"p://images.clipartof.com/thumbnails/1050055- Royalty- Free- RF- Clip- Art- Illustra*on- Of- A- S*nky- Pile- Of- Poop- Character.jpg Flag: h"p://2.bp.blogspot.com/- wqez9n5gp3i/t3nz46mvtni/aaaaaaaaad0/l4dc9fvwj8w/s1600/denmark- flag- 447x298.jpg
Using next generation sequencing (NGS) Microsof 2010 clip art
Methods: Assaying total gene count Low Gene Count (LGC) High Gene Count (HGC) Le Chatelier et al. Nature 500, 541-546 (2013) doi:10.1038/nature12506, Figure 1
Microbial phylum differences in LGC and HGC Le Chatelier et al. Nature 500, 541-546 (2013) doi:10.1038/nature12506, Supplemental Figure 4
Microbial species differences in LGC and HGC Not detected Most abundant Le Chatelier et al. Nature 500, 541-546 (2013) doi:10.1038/nature12506, Figure 2a
LGC Ruminococcus gnavus HGC Faecalibacterium prausnitzii Pro- inflammatory Associated with inflammatory bowel disorder An*- inflammatory Possible probio*c h"p://www.sciencecodex.com/ how_bacteria_with_a_sweet_tooth_may_keep_us_healthy- 121764 h"p://www.processalimentaire.com/var/pa/storage/images/media/images/ faecalibacterium- prausnitzii/65486-1- fre- FR/Faecalibacterium- prausnitzii_ar*cle.jpg
LGC and obesity Le Chatelier et al. Nature 500, 541-546 (2013) doi:10.1038/nature12506 Figure 4
Low Gene Count Obesity
Probiotics h"p://confrazzled.com/the- many- benefits- of- probio*cs/ h"p://www.iherb.com/phillip- s- Colon- Health- Daily- Probio*c- Supplement- Probio*c- Caps- 30- Capsules/31715 Background image: h"p://www.ercprobio*cenzymes.com/files/2393246/uploaded/bacteria.jpg
Caution!! Next Generation Sequencing is not foolproof! Cau*on!
Questions?
Literature Cited 1. Metzker, M. L. (2009). Sequencing technologies the next generation. Nature Reviews Genetics, 11, page 31-46. doi:10.1038/ nrg2626 2. Le Chatelier, E. et al. Richness of human gut microbiome correlates with metabolic markers. Nature 500, 541-546 (2013). 3. Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027 1031 (2006). 4. Sokol, H. et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc. Natl Acad. Sci. USA 105, 16731 16736 (2008). 5. Png, C. W. et al. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am. J. Gastroenterol. 105, 2420 2428 (2010).