Appendix - 2qgnA

1. 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”
2. 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 

3. copy all the accession number, annotation and so on except the actual sequence, save it as “seqh1.blast”
4. By using excel, extract out the accession numbers of “seqh1.blast”
5. 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”
6. another version is “seqh3.blast” (this file only contains the line-by-line listing of the "accession numbers" from the “seqh2.blast”)
7. 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 

8. Construct a multiple sequence alignment and a bootstrap tree on selected homology sequences.
9. Click ALIGNMENT/DO COMPLETE ALIGNMENT to generate multiple alignment using ClustalX.

   Output Guid Tree: newseq.dnd 

   Output Alignment Files: newseq.aln  

10. 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.
11. 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
12. View it in FigTree and take screen snapshot of the tree.