Objective: Continue working with the sequencing data to help decide how to move forward with the project.
Results: Found that the highest likely candidate for the sequences may have been a bacteria Rhodopseudomonas palustris so contacted Dr. Carrie Harwood in the harwood labs at UW medical center to learn more information about that bacteria and see if it was a possible candidate and to see if she had some available DNA that I may be able to work with. I was considering to PCR the DNA with my primers to see if they showed any expression and possible the same I was seeing in my own PCRs. She replied back saying that it was not marine bacteria so it's not likely what I have been sequencing. She also said she should have some DNA on file that I could work with but Dr. Roberts felt that it was not necessary since the species was not marine. Dr. Roberts has me convert all of my sequences into a Fasta file which was uploaded to eagle. (http://eagle.fish.washington.edu/scaphapoda/index.php?dir=Jonathan%2F)
Friday, August 28, 2015
Wednesday, August 12, 2015
Sequencing work and NCBI blasting of new sequences and outliers
1. Take the new data and work with NCBI to try to find out what was sequenced using BlastN and BlastX.
2. Align the new sequences produced on august 10th to the Renilla reniformis sequence used to create primers.
3. Check NCBI blast of sequence outliers that did not align with the majority of sequences using BlastN and BlastX.
4. Check NCBI to see if Renilla sequence matches any other luciferase sequences using BlastN and BlastX.
Results:
1. The concencous sequence did show a relationship to Renilla reniformis though it had a low e-value
2. The alignment of the new sequences to Renilla reniformis only aligned when the cost matrix of the Geneious alignment was at 51%, and when I did a muscle alignment, though it showed a lot of gaps which questions the validity of the alignment. At 51% gaps were visible but were small usually only 1 base pair long.
3. Outliers did not show a strong e-value so unlikely that it would be useable
4. The Renilla sequence used to produce the primers did relate to other Luciferase sequences but with a very low e-value.
Side note: Blogger is not allowing me to add in titles between pictures. Not sure how to work with it so I can better differentiate each area. The conflict seems to be how to images are formatted in blogger
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| NCBI BLASTN SUITE: Consensus sequence produced august 10th 2015 |
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| BLASTX: Consensus sequence produced august 10th 2015 |
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| Geneious alignment of sequences produced august 10th 2015 compared to Renilla reniformis sequence used to produce primers , Cost Matrix 51% |
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| Geneious alignment of sequences produced august 10th 2015 compared to Renilla reniformis sequence used to produce primers, Cost matrix 65% |
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| Geneious alignment of sequences produced august 10th 2015 compared to Renilla reniformis sequence used to produce primers, Cost matrix 70% |
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| Geneious alignment of sequences produced august 10th 2015 compared to Renilla reniformis sequence used to produce primers, Cost Matrix 93% |
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| Muscle alignment of sequences produced august 10th 2015 compared to Renilla reniformis sequence used to produce primers |
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alignment of all sequencing data (note: running blasts on oddball sequences that did not align with majority shown to the left)
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| Sequences blasted |
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| BlastN of PenF-01-luciferase_f.ab1 |
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| BlastX of PenF-01-luciferase_f.ab1 |
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| BlastN of PenF-02-luciferase_f.ab1 |
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| BlastX of PenF-02-luciferase_f.ab1 |
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| BlastN PenR-01-Luciferase_R.ab1 |
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| BlastX PenR-01-Luciferase_R.ab1 |
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| BlastN PenR-02-Luciferase_R.ab1 |
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| BlastX PenR-02-Luciferase_R.ab1 |
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| BlastN SeaPen_luciferase_R1-Rr_46_65F.1.ab1 |
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| BlastX SeaPen_luciferase_R1-Rr_46_65F.1.ab1 |
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| BlastN SeaPen_luciferase_R1-Rr_46_65F.ab1 |
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| BlastX SeaPen_luciferase_R1-Rr_46_65F.ab1 |
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| BlastN of Renilla Luciferase cDNA |
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| BlastX of Renilla Luciferase cDNA |
Monday, August 10, 2015
New sequencing data.
Objective: The last PCR produced on july 22nd was prepared (ethanol precipitation) and sent out for Sanger sequencing. Today I wanted to look at the results and work with them to see if I could align.
Results: The results from the sequencing appear to be successful (shown below) . The HQ percentage is high (90.9, 82.7, 95.1, 96.3) and when aligned all four sequences align well between bases 80-120, 149-523, 569-624. I compared to previous sequences and showed some overlap with previous sequences. The red bars on each side of the sequences are areas that were trimmed due to low quality of the sequence. The top row is a consensus sequence created from the alignment of the for strands It shows the most likely bases when comparing all sequences to each other. With these sequences we should be able to create new primers that will allow me to qPCR my samples to check for gene expression moving forward with my research.
Quality and length of each strand
Alignment of all sequencing data (16 sequences) note: the new strands are the last four that show some overlap with the first 5 sequences.
Genius alignment of the four sequences
Reverse 1 sequnce
Reverse 2 sequence
Forward 1 sequence
Forward 2 sequence
Consensous Sequence:
Results: The results from the sequencing appear to be successful (shown below) . The HQ percentage is high (90.9, 82.7, 95.1, 96.3) and when aligned all four sequences align well between bases 80-120, 149-523, 569-624. I compared to previous sequences and showed some overlap with previous sequences. The red bars on each side of the sequences are areas that were trimmed due to low quality of the sequence. The top row is a consensus sequence created from the alignment of the for strands It shows the most likely bases when comparing all sequences to each other. With these sequences we should be able to create new primers that will allow me to qPCR my samples to check for gene expression moving forward with my research.
Quality and length of each strand
Alignment of all sequencing data (16 sequences) note: the new strands are the last four that show some overlap with the first 5 sequences.
Genius alignment of the four sequences
Reverse 1 sequnce
Monday, August 3, 2015
Behavior graphical represenations and some good molecular news
Objective: Convert data taken from the sea pen videos and create a graphical interpretation of the behavior.
Results:
The graphs ( shown below) represent a total count of hours of behavior throughout a 21 days period, and following graphs cover a 24 hour period the bars represent 3 types of behavior. Extended is when the animal was inflated, Unextended represents when the animal was deflated, and Wave motion is when a peristaltic type movement that ran the length of the animal was visible. If wave motion was visible at any time through the hour it was recorded so when wave motion is shown it does not mean that wave motion occurred for the entire hour it was just visible at some time during that hour. The areas of the graph that do not show any bars means that the animal was not visible at any point during that hour. Some days due to lighting or poor visibility no data points were recorded for the entire day. Later on in the experiment the lights were lowered making it very difficult to see the animals. At one point during the experiment the camera had been hacked so UW I.T. shut down the feed making it impossible to record those days. After 11/24/2014 the light cycle was also changed so data usually starts around 10:00am. It may be useful to try to get more data to look for trends but I will speak with Dr. Roberts to see if I should collect from more videos.
Notes: The sequencing of previous PCR was sent out for Sanger sequencing and received back. Sam said that it was a success and we will definitely be able to use the results. Will go over it with him later this week, and possible be able to move forward with the Molecular experiment.
Data locations:
Behavior Excel Spreadsheet: https://drive.google.com/file/d/0B9noISVg7C3eQ2tZOGpCUEVwSHM/view?usp=sharing
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