 Dear students, in this module I am going to summarize the use of BLAST towards searching nucleotide or amino acid sequences from the databases. You already know now that BLAST can help you to quickly search and align your nucleotide or amino acid sequence by looking at the sequences in the database. So it is an online tool that is available for free and you can input your IDs of proteins or genes and arrive at the comparisons from the databases. There are multiple types of BLAST which you can use to compare these sequences. So the steps, the important steps for BLAST, so step number one, so you have to obtain a query sequence. You remember to perform BLAST you need two things. The first is the query sequence and the second is the database or simply the DB. So how do you get the query sequence? So to find that you first need to know if you have a nucleotide or an amino acid. So by looking at what kind of sequence you have you select the specific protein or amino acid by selecting this drop down list box at the NCBI website. So if the sequence is known you can go to NCBI or others but if the sequence that you have is unknown or you don't have that sequence with you you can use NGS to sequence the gene or mass spectrometry to sequence the proteins. Okay so once you have obtained the sequence using either the experimental methods that is NGS or mass spec then you can input the sequence later or you can obtain the ID by looking at the NCBI website and searching for the specific sequence. In the second step you choose the type of BLAST that you want to have. So you have the query and the DB and you can compare them but it depends on how you want to compare either a nucleotide or a protein. Similarly at the database side you can also have a nucleotide database or a protein database. How do you want to compare that will determine your type of BLAST. So there are several types as you already know if you have two nucleotides that you want to sequence then you can use the nucleotide BLAST. If you have two protein sequences then you can use the protein BLAST or if you want to interchange between the sequences on the query and the DB side then you can look at BLASTX, TBLASTN, TBLASTX and several others as well and you can perform a BLAST. So to summarize the BLASTN is a nucleotide BLAST, the BLASTP is a protein BLAST, the BLASTX is a translated BLAST with nucleotide sequence on the query side and the protein sequence on the other side that is the database side. You have the TBLASTN and the TBLASTX so both are translated BLAST as you can see here with the protein or a nucleotide sequence on the database side respectively. To help you understand the importance of ORF so the BLASTN or nucleotide BLAST has a DNA sequence on both sides, the BLASTP has protein sequence on both sides but the BLASTX has a DNA sequence on this side and a protein sequence on that side on the database side. So since the DNA sequence can have six open reading frames so you need to create six different comparisons with the protein database. So in this way this one BLASTX has a computational cost of six BLASTNs or BLASTPs. If you want to use TBLASTN or TBLASTX so the number of comparison again for TBLASTX is six ORFs on this side, six ORFs on this side so six into six that is equal to 36. So 36 comparisons are made for one TBLASTX. Next you set up your search parameters by looking at the databases and the organisms and so on and then you press the BLAST button here. So the results that are output for you will be the sequences like that which are matching and here you can see the legend, the color key for the matches so the red is for greater than or equal to 200 and black is for less than 40. You can also look at the tabulated search results and your query ID, your description and other information is all tabulated for you. And here are your results in the flat text format so you can look at the various IDs of proteins, in this case hemoglobin that have compared very nicely along with the scores and E values. So in conclusion BLAST is an online tool that is freely available and you can compare nucleotide and a minus it sequences from the nucleotide or a minus it sequences in the databases. Also depending on your need you can utilize the type of BLAST that will help you to compare the sequences.