Multiple Sequence Alignments Assemble DNA sequences into a ‘contig’ Identify conserved residues and domains
Multiple Sequence Alignment of Protein Sc: yeastCe: nematode Hs: humanAt: plant Dm: 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
Sequence reads are usually 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
Middle 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