- G T G T A C A C

Transcription

1 Name Student ID.. Sequence alignment 1. Globally align sequence V (GTGTACAC) and sequence W (GTACC) by hand using dynamic programming algorithm. The alignment will be performed based on match premium of 2, mismatch score of -3 and gap penalty of -1. Calculation - G T G T A C A C - G T A C C Answer - G T G T A C A C G T A C C

2 Write down the alignment # Alignment 1 (Yellow path) V G T G T A C A C W - - G T A C - C Alignment 2 (Green and yellow path) V G T G T A C A C W G T - - A C C Alignment 3 (Green, blue and yellow path) V G T G T A C A C W G - - T A C C Calculate the alignment score for the above alignment using match/mismatch score above and an affine gap penalty with gap opening of -5 and gap extension of -2. Please provide both the calculation process and the alignment score. # Because every alignment presents 2 gaps: one carries 2 residues and another carries 1 residue, thus the alignment score of every alignment could be calculated as follows. Score: 2(5) + [-5+2(-2)]+[-5+1(-2)] = = Calculate alignment score of the following alignments using BLOSUM62 with gap opening of 10 and gap extension of 0.5. Also specify the type of alignment. 2.1 FTFTALILLAVAV Alignment type: # Global alignment F--TAL-LLA-AV Alignment score: # (6+5+(7*4))-(10*3)-(0.5*4) = FTALILL-AV Alignment type: # Local alignment FTAL-LLAAV Alignment score: # (6+5+(7*4))-(10*1)-(0.5*1) = Follow steps shown below to align cxea gene of Dictyostelium discoideum AX4 strain to its cdna sequence. 1) Go to the EBI website ( 2

3 2) Under the local alignment section, choose program Water > Nucleotide 3) Copy both DNA and cdna sequence of cxea gene of Dictyostelium discoideum AX4 strain, and then paste on the box as shown below. 4) Take a look at all parameters used for performing pairwise sequence alignment using Smith-Waterman algorithm (local sequence alignment). 3

4 Parameters: Matrix, Gap opening and Gap extension 5) Click submit and wait until you see the result as follows Summary result 6) You can copy the result in a new notepad and save it as a text file (.txt). Based on the alignment result, how many exons does the cxea gene in Dictyostelium discoideum AX4 strain carry? # This sequence contained 2 exons. The alignment result was saved in the file named Ddiscoidium_cxeA_alingment.txt. You can use Aliview to view the alignment 4

5 If you perform this analysis using global sequence alignment, will you obtain the same result as that provided by the local sequence alignment? # The results are the same because the regions containing matched residues are identical. How could you verify that your predicted number of exons is correct? # There are a number of ways you can used to verify the predicted number of exon; for example, you can translate the concatenated exons and compare the translated nucleotide sequence to the protein database sequence, then check whether the most similar protein is the cxea protein or not. 4. Use the pairwise sequence alignment method to align sequence of the E gene of DENV-3 to that of DENV-4. The steps to perform global sequence alignment are shown below. 1) Go to the EBI website ( 2) Under the global alignment section, choose program Needle > Nucleotide 3) Copy DNA sequence of DENV-3 and DENV-4, then paste on the box as shown below. 5

6 4) Take a look at all parameters used for performing pairwise sequence alignment using Needle-W algorithm (global sequence alignment). Parameters: Matrix, Gap opening and Gap extension Extra parameter: End gap penalty 5) Click submit and wait until you see the result as follows. 6

7 Summary result 6) You can copy the result in a new notepad and save it as a text file (.txt). Based on the alignment result, how much are the similarity of the DNA sequences between the E gene of dengue virus serotype 3 (DENV-3) and E gene of dengue virus serotype 4 (DENV-4)? # In case of nucleotide sequence, percent similarity is the same as percent identity which in this case is 65%. How could you evaluate whether this alignment is correct or not? # You can align amino acid sequences in addition to the nucleotide sequence alignment, then compare the amino acid sequence alignment to the translated nucleotide sequence alignment. We have done this task when we practice section no Follow the steps shown below to locate the envelope protein domain Iii on the envelope protein sequence of the dengue virus serotype 3 (DENV-3). 1) Go to the EBI website ( 7

8 2) Under the local alignment section, choose program Water > Protein 3) Copy amino acid sequence of DENV-3 envelope protein and the envelope protein domain Iii, then paste on the box as shown below. 4) Take a look at all parameters used for performing pairwise sequence alignment using Needle-W algorithm (global sequence alignment). 8

9 Parameters: Matrix, Gap opening and Gap extension 5) Click submit and wait until you see the result as follows. Summary result 6) You can copy the result in a new notepad and save it as a text file (.txt). Where is the envelope protein domain Iii on the DENV-3 envelope protein? Please answer this question by stating the amino acid positions on the DENV-3 envelope protein. # Based on the result shown above, the envelope protein domain Iii is located from amino acid 290 to

10 6. Align E gene and envelope protein sequences of dengue virus (DENV-1,2,3 and 4) using G-INS-i in MAFFT. Follow the steps below to align nucleotide sequences. 1) Go to the to download program Aliview. This program will be used to view aligned sequences. Please read install instruction on the website. 10 2) Follow the link here to the MAFFT website.

11 3) Choose to perform Alignment. This function is located on the left-handed side menu as follow. 4) After clicking the Alignment, copy DNA sequences of the DENV-1 to 4 and paste to the box as follows. 5) Set parameter values as follows. Any hidden option/parameter was left as default setting. 11

12 12 6) Click submit. After a while, you will see the alignment result as follows.

13 This alignment is in clustal format. 7) Click Fasta format, then you will see the alignment in fasta format. Copy the alignment and paste on a new notepad, then save the alignment file as a text with extension.txt or.fasta. 8) Open the program Aliview. Click File > Open file, then choose your alignment file. You will see the alignment as follows. 13

14 Click as follows. These steps will translate you DNA sequence to amino acid sequence alignment. Make sure that you choose Standard code and Reading frame ) You can apply these steps to align envelope protein sequences of these dengue viruses. After aligning the protein sequences, save the result in a fasta format. 10) Compare the amino acid sequence alignment obtained from step 9) to the translated nucleotide sequence alignment obtained from step 8). Manually correct your DNA sequence alignment based on the amino acid sequence alignment. How many positions in the DNA sequence alignment do you need to edit in order to correct the DNA sequence alignment? # After comparing amino acid to nucleotide sequence alignment, you would see that you need to move nucleotide from position 471 to 465. The alignment would change from Fig.1 to Fig.2 as follows which after translation, the alignment would look just like the amino acid sequence alignment. Fig. 1 Fig. 2 14

15 7. Apply sequence alignment technique to answer the following questions. Explain how you align sequences by informing what method and options/parameters you have chosen. If the alignment needs to be manually edited, please state that the result obtained from the manually edited alignment. 7.1 How many exons are there in the luciferase gene of Luciola lateralis? # I performed a local pairwise sequence alignment using EMBOSS Water with default parameter values shown as follows. The result showed that this gene contains 7 exons. The alignment result is saved in the file named Luciferase_local_alignment.pdf. 7.2 Where are the conserved regions in the mtcoi gene of fireflies? Until recently, there are six complete mtgenome sequences of fireflies: Asymmetricata circumdata, Bicellonychia lividipennis, Luciola substriata, Aquatica leii, Luciola cruciate, Pyrocoelia rufa, that are available in the GenBank database. The mtcoi sequence of Rhagophthalmus lufengensis was used as an outgroup here. Answer this question by writing down the alignment of the longest conserved region observed in the alignment. # I performed multiple sequence alignment using MAFFT with option Auto and default parameter values. The alignment at the 3 end needs to be edit and the alignment at the 3 end would change from as shown in Fig.1 to that as shown in Fig. 2 Fig. 1 Fig. 2 The longest conserved region is located from position 340 to position 348 as follows. 15