Appendix - 2qgnA
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- The human amino acid sequence fasta file of pdb: 2qgnA tRNA isopentenyltransferase 1 was retrieved from http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=31581534. This is the query sequence and saved as “sequencesh.fasta”
- Conduct BLASTp search against the NCBI non-redundant protein database Version 2.2.10 and save it as “seqh.blast”. This database was downloaded on the Oct-19-2004. Blast sequencesh.fasta query sequence with the non-redundant protein database.
D:\blast\blastall -p blastp -d D:\blast\databases\nr -i sequencesh.fasta -o seqh.blast
- copy all the accession number, annotation and so on except the actual sequence, save it as “seqh1.blast”
- By using excel, extract out the accession numbers of “seqh1.blast”
- since 25 of the 500 matches has E-value of zero which means they are not significant, all others have very small E-values. Some of the similar sequences with nearly identical annotation will be drop out to ease alignment. Saved as “seqh2.xls”
- another version is “seqh3.blast” (this file only contains the line-by-line listing of the "accession numbers" from the “seqh2.blast”)
- Obtain Fasta Format file of the sequences found with blast and save it as “newseqh.fasta”
D:\blast\fastacmd -d D:\blast\databases\nr -i seqh3.blast -o H:\newseqh.fasta
- Construct a multiple sequence alignment and a bootstrap tree on selected homology sequences.
- Click ALIGNMENT/DO COMPLETE ALIGNMENT to generate multiple alignment using ClustalX.
Output Guid Tree: newseq.dnd
Output Alignment Files: newseq.aln
- Click TREES/DRAW NJ TREE to draw neighbour-joining trees.According to this NJ tree, click TREES/BOOTSTRAP NJ TREE and use 100 bootstrap trials; save it as newseqh.ph and newseqh.phb respectively.
- use Webserver that converts sequence identifiers into species names. Load up the newseqh.fasta file with newseqh.phb and save the output as newseqh_names.phb. http://foo.maths.uq.edu.au/~huber/BIOL3004/gi2name.pl
- View it in FigTree and take screen snapshot of the tree.