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

Contig Assembly

ABI Sequencing: Relies on Primer-Directed DNA Synthesis

Chain Terminators are dideoxy NTP’s

H

ABI Sequencing: Relies on Primer-Directed DNA Synthesis

ABI Sequencing

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

Beginning of an ABI read

Beginning of an ABI read

Middle of an ABI read

Middle of an ABI read

End of an ABI Read

End of an ABI Read

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

Protein MSA

• Assemble sequences in fasta format in 1 file

• Prepare multiple sequence alignment (MSA) with ClustalW

• Shade conserved residues using BoxShade

Assemble sequences in fasta format in 1 file

Prepare multiple sequence alignment (MSA) with ClustalW

Shade conserved residues using BoxShade

Protein MSA

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

Modify BoxShade Output in MS Word

In Class MSA Tutorial

• Assemble sequences into a contig using CAP

• Create a MSA of protein sequences for use in PowerPoint