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

2. Multiple Sequence Alignment of Protein Sc: yeastCe: nematode Hs: humanAt: 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. 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

11. Middle of an ABI read

13. 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