multiple sequence alignments assemble dna sequences into a ‘contig’ identify conserved...
TRANSCRIPT
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Multiple Sequence Alignments
Assemble DNA sequences into a ‘contig’ Identify conserved residues
and domains
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Multiple Sequence Alignment of Protein
Sc: yeast Ce: nematodeHs: human At: plantDm: fly
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Contig Assembly
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ABI Sequencing: Relies on Primer-Directed DNA Synthesis
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Chain Terminators are dideoxy NTP’s
H
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ABI Sequencing: Relies on Primer-Directed DNA Synthesis
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ABI Sequencing
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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
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Beginning of an ABI read
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Beginning of an ABI read
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Middle of an ABI read
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Middle of an ABI read
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End of an ABI Read
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End of an ABI Read
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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
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Protein MSA
• Assemble sequences in fasta format in 1 file
• Prepare multiple sequence alignment (MSA) with ClustalW
• Shade conserved residues using BoxShade
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Assemble sequences in fasta format in 1 file
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Prepare multiple sequence alignment (MSA) with ClustalW
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Shade conserved residues using BoxShade
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Protein MSA
• Modify BoxShade Output for use – in MS Word doc– in PowerPoint presentation– in web page
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Modify BoxShade Output in MS Word
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In Class MSA Tutorial
• Assemble sequences into a contig using CAP
• Create a MSA of protein sequences for use in PowerPoint