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Published byWilfred Stewart Modified over 9 years ago
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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: yeastCe: nematode Hs: humanAt: plant Dm: 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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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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Middle 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
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