Sequence Obtained Template Assessment Answers

BI2234 coursework 3 template. Insert your answers into the spaces below. Remember to use courier new font for sequences. The completed template should be submitted as both a word file and a pdf.
Task 1. Paste the edited sequence obtained from the file GeneX-SeqM13F1 into the space below in FASTA format.
gt41596_F1P_R2NNNNNNNNNNNNNNNNNNNNNGANGGGCACGGTGAGCCTGCGGAGTCCTCAGACATGCTGGGGCTNNNNNNNNCAGAGTGACACTTCTCCTGTTCTTCTGTCATCTTCAGGTAAGGGTCGAGGAGATTCATGCGAAAGAATTAAAGAACAGCTTCTCGCAGTAAAAAAAGAAAGAAAATCCTTCTTTCTAAATCCAAGTGCTGTGCCCAAAGACTGCAGTGACAGTCTGTGAGGGCGCACAGCTTGACTGACAAACTTGAATCCTTTAAAGTTAAAAAAAACAACAAAAAACAACAAAATGCCAAAAATAAAAGTTTTTTGGTCCTTCGACTTTTCACAATTTCGCAGGCGAGAATGCAAACAGCAAATGATCGCTTTCTAATGCTTAGCTTTGAAACTGTGACTTTAAAGAGAAATGTGACTGAGAGGTCTGCTCTTGGTGGATTGCTGAAGAGTCTACGGGCTCAGGAGGCGATGCCTGGATTTATGTTTGACGGCAAGGAAATGATTGGGGGGAACTTTTTGTAGGCGGAGACGTAATATGACTCTGCTCTAAAGAACTCTTGCAGTCAACAACAGTGAAAATCCAACTCAAGTAGGAAAAAGGTGTTTTTCCCCCTTATTATCTTTGGTTTCAAGTAAATGTTAAGCATGGCTTAAAGTTGAAAAAGTTAGGTTGAATTAAAAGTTGCATTTTACACTTTCTGAGAAAAATAATGGAAGTAAGACTATAATTACTAATCTTACCTTACAGTAATGTGAACCATTTTAATATTATACTGATATCGAGGGAAATAGAAAATATACCAACCCTAGGAATTTTCTTTGAGGACAAACACAAAGTCCGTATTANCNNNNNNTNNAANATTCAACAAANNTGTTAGAATTATAAACACATTGNAGTTCTTCTAGCTTTANTATACTTGCTTANAGNCTTTTTTTCTTTTATTCCNTATTTTACAGAAATAAAAAGGNTANNTTNCATTTTTATACAACTTGTCCCGCTNNNAANNGTANTTTGNNNATGTGNNNGTGAANGCNAANTNGANGANNTTTACNNCTNNNGNATNNNNNNNNTCNNTTCNNNNNNNNNNANNTNNNNNNNNNNNNGNNNANNNNNANNNNNGNNNNNNNNNNANNNNNNNNNNNNTTNNNNNNNNNNNTask 2. Paste your assembled sequence (contig) into the space below in FASTA format (The contig can be assembled manually or with CAP3).
gtContig1NNNNNNNNNNNNNNCNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNTNNNNNNNNNNNTTNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNTNNNNNTNGNNNNNNNNNNTNNNNNNNNNNTNANTTTNNCNGTNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNTGNNNNNNNATTTNTTNTTTTTTANTGNNNNGNAGNNNNTNNTNATNNTTTNNCANGAATNNNTCGNNNNTNNNGANGATGNCNGANNNNCAGGNNNAGNNTCANTNNGACGNTNCCAGCCCCAGCATGTNNGAGGACTCNNCAGGCTCACCGTGCCCGTCCGGGTCCGGTTCGGACACGGAGAACACCCGNCCGTCCGACANCCACCTCCTTTTGGGTCCAGACTACAAGAAGGAGGGNGAAGAAGAAAAGTTCCCCGTGTGTATCAGAGATGCGGTGTCCCAGGTGTTGAAGGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTTAACGGCTCGACTAAAAGCAAACCTCACGTCAAAAGACCCATGAACGCGTTCATGGTCTGGGCTCAGGCTGCACGGAGGAAGCTTGCCGATCAATACCCGCATCTGCACAACGCAGAACTCAGCAAAACCCTGGGCAAACTTTGGAGGTAGGTTCGCTTTGCATTTCCAATCACTTTTTTCAGTGCACTTTTACGCGCCGCTGTGCGCAGCGCACTATTTCTTCGATGAAATGCACCACCTGACTGCTGTTTCAAGTAGAGCTTTACAGTCTGGGAACATGACTAATGCATTTCAAATTATATTTCCCTCTTTTTTATTGTGATTCTGAATACACTTCTTATTCCCATGCCTGAGTGCATGCACAGAAGACTGTAGGCTACGCAAACTTTTATGTATCTGCTTGTTGTTGATTAGTTCTATTAAAACAATACAATGATTCAGGAAATGTCACATCTCTTGCACTAATTAATCACCCGTGTTTTGTGCTTTTTGGTAATTGAATGCATTTTAATTTGAATGTACAATTGTCTGTGAATTTTCAGATTGCTCAACGAAGTAGAGAAGCGTCCGTTTGTAGAAGAAGCTGAGCGTTTGAGGGTCCAGCACAAGAAGGACCATCCCGACTACAAGTATCAGCCAAGGCGGAGAAAATCTGTCAAGAACGGGCAAAACGATCCCGAGGAAGGCGAGCAAACCCACATCTCTCCAAATGCGATCTTCAAGGCGCTGCAGCAGGCCGATTCTCCAGCGTCTAGCCTGGGCGAGGTGCACTCTCCAGGAGAGCACTCAGGTAGAAAAGAANTTTAATCAAATCNTTAAAATAAAAGGAATTTCTAGTTNGNTNGNTTTTTTCAAAANCGACTNATTTTGGGNCNNNTTNGNAAAATask 2. Paste your assembled and numbered sequence (contig) into the space below. The sequence should also indicate intron-exon boundaries identified in task 4.
Number of segment pairs 42 number of pairwise comparisons 6
means given segment - means reverse complement
Overlaps Containments No. of Constraints Supporting Overlap
Contig 1
41478_F1p2_R-
MG-FIPpGEMT7-S95
Task 2. Insert a scale diagram in the space below to show how the seven individual sequences have been assembled into a contig.
DETAILED DISPLAY OF CONTIGS Contig 1
. . . . . .
41478_F1p2_R- NNNNNNNNNNNNNNCNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNTNNN ____________________________________________________________
consensus NNNNNNNNNNNNNNCNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNANNNTNNN . . . . . .
41478_F1p2_R- NNNNNNNNTTNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNTNNNNNTNGNNNNNNNN ____________________________________________________________
consensus NNNNNNNNTTNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNTNNNNNTNGNNNNNNNN . . . . . .
41478_F1p2_R- NNTNNNNNNNNNNTNANTTTNNCNGTNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ____________________________________________________________
consensus NNTNNNNNNNNNNTNANTTTNNCNGTNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN . . . . . .
41478_F1p2_R- NNGNNNNNNNNNNNNTGNNNNNNNATTTNTTNTTTTTTANTGNNNNGNAGNNNNTNNTNA ____________________________________________________________
consensus NNGNNNNNNNNNNNNTGNNNNNNNATTTNTTNTTTTTTANTGNNNNGNAGNNNNTNNTNA . . . . . .
41478_F1p2_R- TNNTTTNNCANGAATNNNTCGNNNNTNNNGANGATGNCNGANNNNCAGGNNNAGNNTCAN ____________________________________________________________
consensus TNNTTTNNCANGAATNNNTCGNNNNTNNNGANGATGNCNGANNNNCAGGNNNAGNNTCAN . . . . . .
41478_F1p2_R- TNNGACGNTNCCAGCCCCAGCATGTNNGAGGACTCNNCAGGCTCACCGTGCCCGTCCGGG ____________________________________________________________
consensus TNNGACGNTNCCAGCCCCAGCATGTNNGAGGACTCNNCAGGCTCACCGTGCCCGTCCGGG . . . . . .
41478_F1p2_R- TCCGGTTCGGACACGGAGAACACCCGNCCGTCCGACANCCACCTCCTTTTGGGTCCAGAC ____________________________________________________________
consensus TCCGGTTCGGACACGGAGAACACCCGNCCGTCCGACANCCACCTCCTTTTGGGTCCAGAC . . . . . .
41478_F1p2_R- TACAAGAAGGAGGGNGAAGAAGAAAAGTTCCCCGTGTGTATCAGAGATGCGGTGTCCCAG ____________________________________________________________
consensus TACAAGAAGGAGGGNGAAGAAGAAAAGTTCCCCGTGTGTATCAGAGATGCGGTGTCCCAG . . . . . .
41478_F1p2_R- GTGTTGAAGGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTTAACGGCTCGAGTMG-FIPpGEMT7-S95 GCGCGTTAACGGCTCGNCT ____________________________________________________________
consensus GTGTTGAAGGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTTAACGGCTCGACT . . . . . .
41478_F1p2_R- AAAAGCAAACCTCACGTCAAAAGACCCATGAACGCGTTCATGGTCTGGGCTCAGGCTGCAMG-FIPpGEMT7-S95 AAAAGCAAACCTCACGTCAAAAGACCCATGAACGCGTTCATGGTCTGGGCTCAGGCTGCA ____________________________________________________________
consensus AAAAGCAAACCTCACGTCAAAAGACCCATGAACGCGTTCATGGTCTGGGCTCAGGCTGCA . . . . . .
41478_F1p2_R- CGGAGGAAGCTTGCCGATCAATACCCGCATCTGCACAACGCAGAACTCAGCAAAACCCTGMG-FIPpGEMT7-S95 CGGAGGAAGCTTGCCGATCAATACCCGCATCTGCACAACGCAGAACTCAGCAAAACCCTG ____________________________________________________________
consensus CGGAGGAAGCTTGCCGATCAATACCCGCATCTGCACAACGCAGAACTCAGCAAAACCCTG . . . . . .
41478_F1p2_R- GGCAAACTTTGGAGGTAGGTTCGCTTTGCATTTCCAATCACTTTTTTCAGTGCACTTTTAMG-FIPpGEMT7-S95 GGCAAACTTTGGAGGTAGGTTCGCTTTGCATTTCCAATCACTTTTTTCAGTGCACTTTTA ____________________________________________________________
consensus GGCAAACTTTGGAGGTAGGTTCGCTTTGCATTTCCAATCACTTTTTTCAGTGCACTTTTA . . . . . .
41478_F1p2_R- CGCGCCGCTGTGCGCAGCGCACTATTTCTTCGATGAAATGCACCACCTGACTGCTGTTTCMG-FIPpGEMT7-S95 CGCGCCGCTGTGCGCAGCGCACTATTTCTTCGATGAAATGCACCACCTGACTGCTGTTTC ____________________________________________________________
consensus CGCGCCGCTGTGCGCAGCGCACTATTTCTTCGATGAAATGCACCACCTGACTGCTGTTTC . . . . . .
41478_F1p2_R- AAGTAGAGCTTTACAGTCTGGGAACATGACTAATGCATTTCAAATTATATTTCCCTCTTTMG-FIPpGEMT7-S95 AAGTAGAGCTTTACAGTCTGGGAACATGACTAATGCATTTCAAATTATATTTCCCTCTTT ____________________________________________________________
consensus AAGTAGAGCTTTACAGTCTGGGAACATGACTAATGCATTTCAAATTATATTTCCCTCTTT . . . . . .
41478_F1p2_R- TTTATTGTGATTCTGAATACACTTCTTATTCCCATGCCTGAGTGCATGCACAGAAGACTGMG-FIPpGEMT7-S95 TTTATTGTGATTCTGAATACACTTCTTATTCCCATGCCTGAGTGCATGCACAGAAGACTG ____________________________________________________________
consensus TTTATTGTGATTCTGAATACACTTCTTATTCCCATGCCTGAGTGCATGCACAGAAGACTG . . . . . .
41478_F1p2_R- TAGGCTACGCAAACTTTTATGTATCTGCTTGTTGTTGATTAGTTCTATTAAAACAATACAMG-FIPpGEMT7-S95 TAGGCTACGCAAACTTTTATGTATCTGCTTGTTGTTGATTAGTTCTATTAAAACAATACA ____________________________________________________________
consensus TAGGCTACGCAAACTTTTATGTATCTGCTTGTTGTTGATTAGTTCTATTAAAACAATACA . . . . . .
41478_F1p2_R- ATGATTCAGGAAATGTCACATCTCTTGCACTAATTAATCACCCGTGTTTTGTGCTTTTTGMG-FIPpGEMT7-S95 ATGATTCAGGAAATGTCACATCTCTTGCACTAATTAATCACCCGTGTTTTGTGCTTTTTG ____________________________________________________________
consensus ATGATTCAGGAAATGTCACATCTCTTGCACTAATTAATCACCCGTGTTTTGTGCTTTTTG . . . . . .
41478_F1p2_R- GTAATTGAATGCATTTTAATTTGAATGTACAATTGTCTGTGAATTTTCAGATTGCTCAACMG-FIPpGEMT7-S95 GTAATTGAATGCATTTTAATTTGAATGTACAATTGTCTGTGAATTTTCAGATTGCTCAAC ____________________________________________________________
consensus GTAATTGAATGCATTTTAATTTGAATGTACAATTGTCTGTGAATTTTCAGATTGCTCAAC . . . . . .
41478_F1p2_R- GAAGTAGAGAAGCGTCC
MG-FIPpGEMT7-S95 GAAGTAGAGAAGCGTCCGTTTGTAGAAGAAGCTGAGCGTTTGAGGGTCCAGCACAAGAAG ____________________________________________________________
consensus GAAGTAGAGAAGCGTCCGTTTGTAGAAGAAGCTGAGCGTTTGAGGGTCCAGCACAAGAAG . . . . . .
MG-FIPpGEMT7-S95 GACCATCCCGACTACAAGTATCAGCCAAGGCGGAGAAAATCTGTCAAGAACGGGCAAAAC ____________________________________________________________
consensus GACCATCCCGACTACAAGTATCAGCCAAGGCGGAGAAAATCTGTCAAGAACGGGCAAAAC . . . . . .
MG-FIPpGEMT7-S95 GATCCCGAGGAAGGCGAGCAAACCCACATCTCTCCAAATGCGATCTTCAAGGCGCTGCAG ____________________________________________________________
consensus GATCCCGAGGAAGGCGAGCAAACCCACATCTCTCCAAATGCGATCTTCAAGGCGCTGCAG . . . . . .
MG-FIPpGEMT7-S95 CAGGCCGATTCTCCAGCGTCTAGCCTGGGCGAGGTGCACTCTCCAGGAGAGCACTCAGGT ____________________________________________________________
consensus CAGGCCGATTCTCCAGCGTCTAGCCTGGGCGAGGTGCACTCTCCAGGAGAGCACTCAGGT . . . . . .
MG-FIPpGEMT7-S95 AGAAAAGAANTTTAATCAAATCNTTAAAATAAAAGGAATTTCTAGTTNGNTNGNTTTTTT ____________________________________________________________
consensus AGAAAAGAANTTTAATCAAATCNTTAAAATAAAAGGAATTTCTAGTTNGNTNGNTTTTTT . . . . . .
MG-FIPpGEMT7-S95 CAAAANCGACTNATTTTGGGNCNNNTTNGNAAAA ____________________________________________________________
consensus CAAAANCGACTNATTTTGGGNCNNNTTNGNAAAATask 2. Give a brief account of the strategy used to assemble the contig shown above. Discuss any problems that were encountered and how these problems were overcome.
The contigs were assembled using CAP3 program. The sequences were merged as a single fasta file and uploaded into CAP3. The results were displayed after processing. The program removes 5 and 3 ends and constructs contigs by MSATask 3. Give the identity of gene X and briefly explain how you reached your conclusion.
The contig was blasted and the first hit was Cottoperca gobio genome assembly, chromosome 8 with 70 query coverage. Hence the identity of the gene is Cottoperca gobio genome assembly, chromosome 8Task 4. In the space below, paste the region corresponding to the second exon of the alignment of the sea bass gene X contig with gene X mRNA from Epinephelus coioides, Include a few nucleotides of flanking sequence from the contig. Also go back to the numbered sequence generated in task 2 (ii) and indicate intron-exon boundaries by converting the introns to lower case.
Second exon
ATTTGAATGTACAATTGTCTGTGAATTTTCAGATTGCTCAACGAAGTAGAGAAGCGTCCGTTTGTAGAAGAAGCTGAGCGTTTGAGGGTCCAGCACAAGAAGGACCATCCCGACTACAAGTATCAGCCAAGGCGGAGAAAATCTGTCAAGAACGGGCAAAACGATCCCGAGGAAGGCGAGCAAACCCACATCTCTCCAAATGCGATCTTCAAGGCGCTGCAGCAGGCCGATTCTCCAGCGTCTAGCCTGGGCGAGGIntron exon boundaries
nnnnnnnnnnnnnncnannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnannntnnnnnnnnnnnttnncnnnnnnnnnnnnnnnnnnnnnnnnnnntnntnnnnntngnnnnnnnnnntnnnnnnnnnntnantttnncngtnannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnngnnnnnnnnnnnntgnnnnnnnatttnttnttttttantgnnnngnagnnnntnntnatnntttnncangaatnnntcgnnnnTNNGACGNTNCCAGCCCCAGCATGTNNGAGGACTCNNCAGGCTCACCGTGCCCGTCCGGGTCCGGTTCGGACACGGAGAACACCCGNCCGTCCGACANCCACCTCCTTTTGGGTCCAGACTACAAGAAGGAGGGNGAAGAAGAAAAGTTCCCCGTGTGTATCAGAGATGCGGTGTCCCAGGTGTTGAAGGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTTAACGGCTCGACTAAAAGCAAACCTCACGTCAAAAGACCCATGAACGCGTTCATGGTCTGGGCTCAGGCTGCACGGAGGAAGCTTGCCGATCAATACCCGCATCTGCACAACGCAGAACTCAGCAAAACCCTGGGCAAACTTTGGAGGTAGGTTCGCTTTGCATTTtgcactctccaggagagcactcaggtagaaaagaantttaatcaaatcnttaaaataaaaggaatttctagttngntngnttttttcaaaancgactnattttgggncnnnttngnaaaaATTTGAATGTACAATTGTCTGTGAATTTTCAGATTGCTCAACGAAGTAGAGAAGCGTCCGTTTGTAGAAGAAGCTGAGCGTTTGAGGGTCCAGCACAAGAAGGACCATCCCGACTACAAGTATCAGCCAAGGCGGAGAAAATCTGTCAAGAACGGGCAAAACGATCCCGAGGAAGGCGAGCAAACCCACATCTCTCCAAATGCGATCTTCAAGGCGCTGCAGCAGGCCGATTCTCCAGCGTCTAGCCTGGGCGAGGTask 5. Briefly assess whether the annotation of gene X on the sea bass genome browser supports your identification of intron-exon boundaries from task 4
Yes the annotation supports the boundary regions
Task 5. Zoom in to the 40 or so nucleotides that span the beginning of the second exon and paste the image of the browser window in the space below

Task 5. Assess the accuracy of the annotation of the gene X paralogue (second-best BLAT hit).
Larimichthys crocea genome assembly, chromosome VIII is the second best blast result
Task 6. Paste an alignment of the protein sequence of human gene X with the sea bass gene X and its paralogue. This should also (i) show the location of the DNA binding domain and (ii) amino acids that are altered as a result of the disease associated mutations found in task 7.
Sea bass sequence is paralogous with human SOX 9 sequence.
Alignment statistics for match 1
Score Expect Identities Gaps Strand
248 bits(134) 8e-63 290/369(79) 7/369(1) Plus/Plus
Query 311 CCAGCCCCAGCATGTNNGAGGACTCNNCAGGCTCACCGTGCCCGTCCGGGTCCGGTTCGG 370

Sbjct 437 CCAGCCCCACCATGTCCGAGGACTCCGCGGGCTCGCCCTGCCCGTCGGGCTCCGGCTCGG 496
Query 371 ACACGGAGAACACCCGNCCGTCCGACANCCAC-CT-CCTTTTGG-G-TCCAGA-CTACAA 425

Sbjct 497 ACACCGAGAACACGCGGCCCCAGGAGA-ACACGTTCCCCAAGGGCGAGCCCGATCT-GAA 554
Query 426 GAAGGAGGGNGAAGAAGAAAAGTTCCCCGTGTGTATCAGAGATGCGGTGTCCCAGGTGTT 485

Sbjct 555 GAAGGAGAGCGAGGAGGACAAGTTCCCCGTGTGCATCCGCGAGGCGGTCAGCCAGGTGCT 614
Query 486 GAAGGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTTAACGGCTCGACTAAAAG 545

Sbjct 615 CAAAGGCTACGACTGGACGCTGGTGCCCATGCCGGTGCGCGTCAACGGCTCCAGCAAGAA 674
Query 546 CAAACCTCACGTCAAAAGACCCATGAACGCGTTCATGGTCTGGGCTCAGGCTGCACGGAG 605

Sbjct 675 CAAGCCGCACGTCAAGCGGCCCATGAACGCCTTCATGGTGTGGGCGCAGGCGGCGCGCAG 734
Query 606 GAAGCTTGCCGATCAATACCCGCATCTGCACAACGCAGAACTCAGCAAAACCCTGGGCAA 665

Sbjct 735 GAAGCTCGCGGACCAGTACCCGCACTTGCACAACGCCGAGCTCAGCAAGACGCTGGGCAA 794
Query 666 ACTTTGGAG 674

Sbjct 795 GCTCTGGAG 803
Task 6. Insert a table listing the genes that flank gene X and its paralogue in sea bass and the chromosomal location of the corresponding genes, and gene X itself, in humans.
Flanking genes- MYL6P5 and CASC17Paralogs in sea bass- none found by blast
Task 6. Discuss the relationships between the human and sea bass sequences in the light of the results above from task 6.
Paralogous sequences have not been found in humans and sea bass flanking genes. Hence it can be concluded that the sequences are not evolutionarily related together.
Task 7. Comment on the impact of disease-associated mutations on the function of the protein encoded by gene X and briefly discuss the genetic and phenotypic aspects of the associated disease. Remember to indicate the site of the altered amino acids on your multi-sequence alignment from task 6 above.
As per the alignment 292/371 of amino acids aligned with eachother. The remaining mutations mainly affect cancer susceptibility and DNA binding
Task 8. Insert a detailed flow diagram showing a strategy to express gene X in E. coli using the vector pET-21a. The diagram should include the key steps and any specialised reagents.
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References

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