High-Throughput Sequencing
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Talk on High-Throughput Sequencing: Overview and Selected Applications for Masters students. Nov 9th 2011
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High-Throughput Sequencing
- 7. 454 Life Sciences (Roche)
- 8. 454 Life Sciences (Roche)
- 10. SOLiD Sequencing Requires emPCR Long run-times Short read-lengths (stuck at 50bp) Sequences in colour space
- 11. Source: http://www.politigenomics.com/next-generation-sequencing-informatics *Now improved to 1 kb reads and choice of 3, 8 or 20 kb inserts #b=bases, B=bytes Vendor: Roche Illumina ABI Technology: 454 Solexa GA SOLiD Platform: GS20 FLX Ti I II IIx 1 2 3 Reads: (M) 0.5 0.5 1.25 28 100 150 40 115 320 Fragment Read length: 100 200 400* 35 50 100 25 35 50 Run time: (d) 0.25 0.3 0.4 3 3 5 6 5 8 Yield: (Gb#) 0.05 0.1 0.5 1 5 15 1 4 16 Rate: (Gb/d) 0.2 0.33 1.25 0.33 1.67 3 0.34 1.6 2 Images: (TB#) 0.01 0.01 0.03 0.5 1.1 2.8 1.8 2.5 1.9 Paired-end Read length: 200 400 2×35 2×50 2×100 2×25 2×35 2×50 Insert: (kb) 3.5 3.5* 0.2 0.2 0.2 3 3 3 Run time: (d) 0.3 0.4 6 10 10 12 10 16 Yield: (Gb) 0.1 0.5 2 9 30 2 8 32 Moore’s law applies! The Sequencing Singularity! Everything published is out of date!
- 15. “ De novo assembly” versus “ alignment against template” (aka “re-sequencing”)
- 17. The Birth of Genomic Epidemiology for Bacteria
- 18. The Birth of Genomic Epidemiology for Bacteria
- 19. Sequencing in Birmingham @mjpallen @pathogenomenick #AAMTHI
- 25. Outbreak isolates distinguishable at only three loci SNP 1 SNP 2 SNP 3 AB0057 C A G M1 C A G M2 T A G M3 T A T M4 T A G C1 T T G C2 T A G
- 31. Ion Torrent Millions of wells reading sequences Microchip detects release of protons ~3 hour run-time ~£500 cost per run
- 37. Malignant Darwinism Mutational frequency heterogeneity analysis to become an integral component of molecular pathology Cancer is an evolutionary process
- 39. Applications: Cancer Biology Deep precision measurements of mutation frequency in a tissue can be made using next generation sequencing of PCR amplicons spanning the mutation
- 46. Multiple Displacement Amplification Single-cell Genomics Or FACS or dilution or microfluidics)
- 48. What will you do when you can sequence everything?
Editor's Notes
- Note this is a new version of the slide I gave you a few weeks ago – it has been updated; I’ve also taken out the data components which made it too busy
- Are there others we should mention? Also need to flag up newly realised strengths e.g. power of paired end reads to generate single contig of a bacterial chromosome
- Again your comments much valued here!
- 95 & 63 strains
- 95 & 63 strains
- Change shading Ultimately, we detected three SNP loci which allowed us to distinguish between isolates in the outbreak. These are presented with reference to an unrelated Acinetobacter reference, AB57 which we consider has the “ancestral” state at these loci.
- Referring back to the outbreak diagram we can plot these consequent genotypes onto each isolate. So when considering C1 it can be seen that it has a unique genotype compared with the others thus making it hard to make a compelling case for transmission from any of the military patients. But when we consider the case of C2 it can be seen that it shares the same genotype as M2 and M4. Given that M2 and C2 were in neighboring beds around week 4 but M4 did not come into contact with C2 at any point, we believe we can make a strong case for transmission from M2 and C2.
- You can read the full story of these study in the Journal of Hospital Infection where it is available as an online pre-print. Our analyses support transmission of MDR-Aci from the wound of a military patient M2 to the respiratory tract of a civilian patient C2. As MDR-Aci was not isolated from C2 until several weeks after M2 left the adjacent bed, however, we cannot determine when and how transmission occurred. One possibility is that C2 became colonised when the two patients were nursed together, but that colonisation did not reach detectable levels in the sputum until much later. Another possibility is that M2 contaminated the local environment and C2 acquired the organism from the environment only after M2 had left the ward. This latter option would be consistent with a significant role of the environment.