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Eisen.CentralValley2024.pdf
The document outlines innovations in sequencing and bioinformatics, specifically focusing on sequence-based surveillance workflows. It discusses various sequencing technologies, sample preparation methods, and key issues such as cost, accuracy, and efficiency. Additionally, it highlights the evolution of sequencing methods from manual to automated processes and emphasizes the importance of informatics in analyzing genetic data.
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Eisen.CentralValley2024.pdf
- 1. Innovations in Sequencing& Bioinformatics Talk for Healthy Central Valley Together Research Workshop Jonathan A. Eisen University of California, Davis January 31, 2024 linktr.ee/jonathaneisen
- 2. Sequence Based SurveillanceWorkflow Sequence IFX
- 3. Sequence IFX DNA RNA Amplicon Shotgun Target Enrichment Sequence BasedSurveillance Workflow
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- 7. Key Issues • Cost/ bp • Read length • Output per run • Paired end • Ease of feeding • Error profiles and accuracy • Barcoding potential • Detection of modifications • Sensitivity to low quality DNA/RNA • RNA, DNA, or mixed RNA/DNA 12 12
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- 9. Evolution of Sequencing Gen0: Proto Sequencing
- 10. Evolution of Sequencing Gen0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing
- 11. Evolution of Sequencing Gen0: Proto Sequencing Sanger Maxam-Gilbert Gen 1: Manual Sequencing Gen 2: Automated Sanger
- 12. Evolution of Sequencing Gen0: Proto Sequencing Sanger Maxam-Gilbert ABI-Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones
- 13. Oxford NanoPore Pacbio Helicos Evolution of Sequencing Gen0: Proto Sequencing Sanger Maxam-Gilbert ABI-Solid Solexa- Illumina 454- Roche Ion Torrent Gen 1: Manual Sequencing Gen 2: Automated Sanger Gen 3: Clusters Not Clones Gen 4: Single Molecule
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- 17. Multiplex PCR vsCapture https://www.celemics.com/resources/blogs/target-capture-multiplex-pcr-vs-hybridization-capture/
- 18. Capture Methods 18 High throughputsample preparation Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput Nature Methods, 2010, 7: 111-118 RainDance Microdroplet PCR Roche Nimblegen Salid-phase capture with custom- designed oligonucleotide microarray Reported 84% of capture efficiency Reported 65-90% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high-throughput-equencing 18
- 19. High throughput samplepreparation Sample preparation Clusters amplification Sequencing by synthesis Analysis pipeline Introduction Illumina GAII High throughput Agilent SureSelect Solution-phase capture with streptavidin-coated magnetic beads Reported 60-80% of capture efficiency From Slideshare presentation of Cosentino Cristian http://www.slideshare.net/cosentia/high- throughput-equencing Capture Methods 19 19
- 20. Small Amounts ofDNA 20 http://www.epibio.com/docs/default-source/protocols/nextera-dna-sample-prep-kit-(illumina--compatible).pdf?sfvrsn=4 20
- 21. HiC 21 Suggested by CarlosBustamante and Keith Bradnam 21
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- 23. Single Cell Sequencing 23 Suggestedby Keith Robison 23
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- 25. Library Prep Questions •Specific targets? • Taxa? • Genes (e.g., antibiotic resistance) • How many targets? • Total community analysis? • Looking for novel taxa or subtypes? • Looking for novel genes? (e.g., drug resistance)
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- 30. Shotgun Sequencing DNA inputs offixed carbon or nitrogen from external sources. As with Leptospirillum group I, both Leptospirillum group II and III have the genes needed to fix carbon by means of the Calvin–Benson– Bassham cycle (using type II ribulose 1,5-bisphosphate carboxy- lase–oxygenase). All genomes recovered from the AMD system contain formate hydrogenlyase complexes. These, in combination with carbon monoxide dehydrogenase, may be used for carbon fixation via the reductive acetyl coenzyme A (acetyl-CoA) pathway by some, or all, organisms. Given the large number of ABC-type sugar and amino acid transporters encoded in the Ferroplasma type Figure 4 Cell metabolic cartoons constructed from the annotation of 2,180 ORFs identified in the Leptospirillum group II genome (63% with putative assigned function) and 1,931 ORFs in the Ferroplasma type II genome (58% with assigned function). The cell cartoons are shown within a biofilm that is attached to the surface of an acid mine drainage stream (viewed in cross-section). Tight coupling between ferrous iron oxidation, pyrite dissolution and acid generation is indicated. Rubisco, ribulose 1,5-bisphosphate carboxylase–oxygenase. THF, tetrahydrofolate. articles NATURE | doi:10.1038/nature02340 | www.nature.com/nature 5 ©2004 NaturePublishing Group
- 31. Issues in Informatics •Compute resources • Remote analysis? • Linking to other data (e.g. clinical, environmental, etc) • Reference data / databases • Need support for FTEs - many “automated” analyses are good but need tuning • Shotgun vs targeted very different
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- 33. Sequence IFX DNA RNA Amplicon Shotgun Target Enrichment Sequence BasedSurveillance Workflow