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Multiple Sequence Alignments

Multiple Sequence Alignments. Assemble DNA sequences into a ‘contig’ Identify conserved residues and domains. Multiple Sequence Alignment of Protein. Sc: yeast Ce: nematode Hs: human At: plant Dm: fly. Contig Assembly. ABI Sequencing: Relies on Primer-Directed DNA Synthesis.

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Multiple Sequence Alignments

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  1. Multiple Sequence Alignments Assemble DNA sequences into a ‘contig’ Identify conserved residues and domains

  2. Multiple Sequence Alignment of Protein Sc: yeast Ce: nematode Hs: human At: plant Dm: fly

  3. Contig Assembly

  4. ABI Sequencing: Relies on Primer-Directed DNA Synthesis

  5. Chain Terminators are dideoxy NTP’s H

  6. ABI Sequencing: Relies on Primer-Directed DNA Synthesis

  7. ABI Sequencing

  8. ABI Sequencing • Sequence reads are usually 600-900 bp in length • Quality of read is poor at beginning and end • Quality is best in the middle of the read

  9. Beginning of an ABI read

  10. Beginning of an ABI read

  11. Middle of an ABI read

  12. Middle of an ABI read

  13. End of an ABI Read

  14. End of an ABI Read

  15. Steps for Contig Assembly • Collect ABI files and assess quality • Trim away ends • Compile into fasta format in 1 file • Assemble contig with ‘CAP’ (Contig Assembly Program) • Evaluate output - more trimming if needed • Repeat CAP assembly if needed • Compare contig with WT or individual reads and make nucleotide assessments

  16. Protein MSA • Assemble sequences in fasta format in 1 file • Prepare multiple sequence alignment (MSA) with ClustalW • Shade conserved residues using BoxShade

  17. Assemble sequences in fasta format in 1 file

  18. Prepare multiple sequence alignment (MSA) with ClustalW

  19. Shade conserved residues using BoxShade

  20. Protein MSA • Modify BoxShade Output for use • in MS Word doc • in PowerPoint presentation • in web page

  21. Modify BoxShade Output in MS Word

  22. In Class MSA Tutorial • Assemble sequences into a contig using CAP • Create a MSA of protein sequences for use in PowerPoint

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