Next Generation Sequencing

Transcription

1 Genomic approaches: Next Generation Sequencing by Sin Ruow Tey and Becky Reese Background image from

2 Genomic sequencing Image by Sin Ruow Tey

3 First generation sequencing: Sanger Source:

4 Source:

5 Sanger sequencing animation

6 Limitations of Sanger sequencing Accuracy: Money: Time:

7 Next Generation Sequencing more accurate cheaper faster Accuracy: Money: Hourglass:

8 Illumina/Solexa STEP 1: Template preparation Source:

9 Source: Illumina/Solexa STEP 2: Attachment

10 Illumina/Solexa STEP 3: Bridge formation Source:

11 Illumina/Solexa STEP 4: Cluster generation Source:

12 Source: Illumina/Solexa STEP 5: Sequencing

13 Source: Illumina/Solexa STEP 6: Imaging

14 Source: Illumina/Solexa STEP 7: Alignment

15 Illumina/Solexa technology animation 10

16 Source: Other NGS platform: Roche 454 Template preparation: emulsion PCR

17 Other NGS platform: Roche 454 Sequencing: pyrosequencing (Roche/454)

18 Other NGS platform: Roche 454 Mapping: de novo assembly Source:

19 Limitations of Illumina/Solexa technology need a reference genome need high coverage to detect SNPs error rate increases past 32bp Hand sign:

20

21 Obesity Often attributed to poor diet Can also be influenced by genetics h"p://cris*naffalcao.wordpress.com/category/obesity/

22 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://

23 Obese microbiome in mice h"p://willpowerisforfatpeople.com/wp- content/uploads/2011/04/fat_mouse.jpg h"p:// content/uploads/2011/09/fat- mouse- skinny- mouse- obesity- longevity.jpg Background image: h"p:// 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, (2006)

24 Le Chatelier et al. Nature 500, (2013) doi: /nature12506

25 Studying the obesity/microbiome connection in humans Dung: h"p://images.clipartof.com/thumbnails/ 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

26 Using next generation sequencing (NGS) Microsof 2010 clip art

27 Methods: Assaying total gene count Low Gene Count (LGC) High Gene Count (HGC) Le Chatelier et al. Nature 500, (2013) doi: /nature12506, Figure 1

28 Microbial phylum differences in LGC and HGC Le Chatelier et al. Nature 500, (2013) doi: /nature12506, Supplemental Figure 4

29 Microbial species differences in LGC and HGC Not detected Most abundant Le Chatelier et al. Nature 500, (2013) doi: /nature12506, Figure 2a

30 LGC Ruminococcus gnavus HGC Faecalibacterium prausnitzii Pro- inflammatory Associated with inflammatory bowel disorder An*- inflammatory Possible probio*c h"p:// how_bacteria_with_a_sweet_tooth_may_keep_us_healthy h"p:// faecalibacterium- prausnitzii/ fre- FR/Faecalibacterium- prausnitzii_ar*cle.jpg

31 LGC and obesity Le Chatelier et al. Nature 500, (2013) doi: /nature12506 Figure 4

32 Low Gene Count Obesity

33 Probiotics h"p://confrazzled.com/the- many- benefits- of- probio*cs/ h"p:// s- Colon- Health- Daily- Probio*c- Supplement- Probio*c- Caps- 30- Capsules/31715 Background image: h"p://

34 Caution!! Next Generation Sequencing is not foolproof! Cau*on!

35 Questions?

36 Literature Cited 1. Metzker, M. L. (2009). Sequencing technologies the next generation. Nature Reviews Genetics, 11, page doi: / nrg Le Chatelier, E. et al. Richness of human gut microbiome correlates with metabolic markers. Nature 500, (2013). 3. Turnbaugh, P. J. et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, (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, (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, (2010).