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Sequencing Technologies: Illumina BIG BIO Juan De la Hoz THANKS - PowerPoint PPT Presentation

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Sequencing Technologies: Illumina BIG BIO Juan De la Hoz THANKS BIG BIO What is Life? What is Life? What is Life? Reproduction Cell division DNA Replication What is Life? Reproduction Cell division DNA Replication

  1. Sequencing Technologies: Illumina BIG BIO Juan De la Hoz

  2. THANKS BIG BIO

  3. What is Life?

  4. What is Life?

  5. What is Life? Reproduction ● Cell division ○ DNA Replication

  6. What is Life? Reproduction ● Cell division ○ DNA Replication https://socratic.org/questions/how-does-dna-replication-relate-to-mitosis

  7. What is Life? Reproduction ● Cell division ○ DNA Replication https://socratic.org/questions/how-does-dna-replication-relate-to-mitosis

  8. DNA Replication https://byjus.com/biology/dna-replication-machinery-enzymes

  9. DNA Replication https://byjus.com/biology/dna-replication-machinery-enzymes

  10. DNA Replication https://byjus.com/biology/dna-replication-machinery-enzymes

  11. DNA Replication https://byjus.com/biology/dna-replication-machinery-enzymes

  12. DNA Replication https://byjus.com/biology/dna-replication-machinery-enzymes

  13. Sequencing by Synthesis

  14. Seq by Synthesis Imagine a small DNA molecule attached to a surface. https://www.cegat.de/en/services/next-generation-sequencing

  15. Seq by Synthesis 1. Add primers https://www.cegat.de/en/services/next-generation-sequencing

  16. Seq by Synthesis 1. Add primers 2. Add free nucleotides 3. Add polymerase https://www.cegat.de/en/services/next-generation-sequencing

  17. Seq by Synthesis 1. Add primers 2. Add free nucleotides 3. Add polymerase 4. Wash 5. Add light and take a photo https://www.cegat.de/en/services/next-generation-sequencing

  18. Seq by Synthesis 1. Add primers 2. Add free nucleotides 3. Add polymerase 4. Wash 5. Add light and take a photo 6. go back to 2 https://www.cegat.de/en/services/next-generation-sequencing

  19. Seq by Synthesis 1. Add primers 2. Add free nucleotides 3. Add polymerase 4. Wash 5. Add light and take a photo 6. go back to 2 https://www.cegat.de/en/services/next-generation-sequencing

  20. Seq by Synthesis errors (1) https://www.cegat.de/en/services/next-generation-sequencing

  21. DNA Replication errors (1): Main driver of evolution https://byjus.com/biology/dna-replication-machinery-enzymes

  22. Seq by Synthesis errors (1) https://www.cegat.de/en/services/next-generation-sequencing

  23. Seq by Synthesis https://www.cegat.de/en/services/next-generation-sequencing

  24. Seq by Synthesis One big Advantage ● Parallelization One big Disadvantage ● Short Reads errors (2) https://www.cegat.de/en/services/next-generation-sequencing

  25. PCR

  26. PCR Signal Intensity can be Exponentially Amplified https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

  27. PCR Signal Intensity can be Exponentially Amplified https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr

  28. PCR Adapters https://www.mclab.com/DNA-Library-Prep-Kit.html

  29. PCR Adapters Unknown Known Known https://www.mclab.com/DNA-Library-Prep-Kit.html

  30. PCR Adapters Cluster formation https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced

  31. PCR errors (1) Adapters Cluster formation https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced

  32. PCR https://kscbioinformatics.wordpress.com/2017/02/13/illumina-sequencing-for-dummies-samples-are-sequenced

  33. Single-end or Paired-end? Fragment size (~500) Read length (150) https://support.illumina.com/bulletins/2016/04/adapter-trimming-why-are-adapter-sequences-trimmed-from-only-the--ends-of-reads.html

  34. RECAP Library preparation Sequencing by synthesis Genomic DNA Extraction Elongation (1nt) Fragmentation Fluorescence + Photo Size selection Repeat Adapter ligation Attach to Plate (paired-end) Bridge PCR Invert molecule Repeat

  35. FASTQ FORMAT

  36. FASTQ FORMAT . . . . . . . . .

  37. FASTQ FORMAT

  38. FASTQ FORMAT

  39. FASTQ FORMAT Quality Score Encoding: Phred+33 (0-41)

  40. FASTQ FORMAT

  41. FASTQ FORMAT

  42. FASTQ FORMAT

  43. FASTQ FORMAT

  44. FASTQ FORMAT

  45. FASTQ FORMAT

  46. FASTQ FORMAT

  47. READ ALIGNMENT Reference Genome https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

  48. READ ALIGNMENT Reference Genome https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

  49. READ ALIGNMENT Reference Genome https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

  50. READ ALIGNMENT Reference Genome https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

  51. READ ALIGNMENT Reference Genome Coverage (1X, 10X, 30X, 100X) https://binf.snipcademy.com/lessons/sequence-file-formats/sam-bam

  52. THANKS BIG BIO

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