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

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

Multiple Sequence Alignments

Assemble DNA sequences into a ‘contig’ Identify conserved residues

and domains

Page 2: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Multiple Sequence Alignment of Protein

Sc: yeast Ce: nematodeHs: human At: plantDm: fly

Page 3: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Contig Assembly

Page 4: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

ABI Sequencing: Relies on Primer-Directed DNA Synthesis

Page 5: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Chain Terminators are dideoxy NTP’s

H

Page 6: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

ABI Sequencing: Relies on Primer-Directed DNA Synthesis

Page 7: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

ABI Sequencing

Page 8: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

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

Page 9: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Beginning of an ABI read

Page 10: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Beginning of an ABI read

Page 11: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Middle of an ABI read

Page 12: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Middle of an ABI read

Page 13: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

End of an ABI Read

Page 14: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

End of an ABI Read

Page 15: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

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

Page 16: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Protein MSA

• Assemble sequences in fasta format in 1 file

• Prepare multiple sequence alignment (MSA) with ClustalW

• Shade conserved residues using BoxShade

Page 17: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Assemble sequences in fasta format in 1 file

Page 18: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Prepare multiple sequence alignment (MSA) with ClustalW

Page 19: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Shade conserved residues using BoxShade

Page 20: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Protein MSA

• Modify BoxShade Output for use – in MS Word doc– in PowerPoint presentation– in web page

Page 21: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

Modify BoxShade Output in MS Word

Page 22: Multiple Sequence Alignments  Assemble DNA sequences into a ‘contig’  Identify conserved residues and domains

In Class MSA Tutorial

• Assemble sequences into a contig using CAP

• Create a MSA of protein sequences for use in PowerPoint