I used DAVID to run gene enrichment on each silo. The input was Uniprot accession codes. Significance cutoffs were at 0.05 for Benjamini corrected p-values (false discovery rate). I’m going to look at the clusters for each silo next.
Biosynthesis of antibiotics and the spliceosome were enriched in all silos, and the spliceosome was enriched in the same place for each silo. Antibiotic synthesis suggests that all of the larvae needed to dedicate large amounts of energy into preventing or fighting infection, possibly suggesting an increased vulnerability, contagion or vial survival or proliferation. This is consistent with mass mortality events commonly seen in hatcheries. An enriched spliceosome could suggest that editing mRNA for translation is occurring at a high rate at this point in development. Could this be because of the larva’s need to produce higher amounts of protein, or could this be the beginning of increased protein synthesis for growth?
Protein folding was the most enriched for every silo. Silo 2 and Silo 3 were almost identical except that Silo 3 lacked enriched oxidation-reduction processes. Silo 9 did have enriched oxidation-reduction pathways as well as intra-golgi vesicle mediated transport which was not enriched in any other silo. I’m not sure how increased intra-golgi vesicle mediated transport might affect the organism. The golgi is responsible for post-translational modification and distribution of proteins. It produces proteoglycans, plays a role in lipid metabolism, and lysosome production. Also, silo 9 lacked enriched viral processes which was enriched in silos 3 and 9 suggesting increased viral influences at cooler silos.
The overall trend is increases in protein production, modification and transport. Silos 2 and 3 have viral processes enriched in addition to those. However, silo 9 has the greatest p-value associated with biosynthesis of antibiotics (9.9E-4 compared to 3.7E-3 for Silo 3 and 1.1E-3 for Silo 2).
- Biosynthesis of antibiotics (9.9E-4)
- Spliceosome (8.4E-4)
- Biological Processes:
- Spliceosome (2.1E-3)
- Biosynthesis of antibiotics (3.7E-3)
- Biological Processes:
- Biosynthesis of Antibiotics (1.1E-3)
- Spliceosome (9.5E-4)
- Biological Processes:
Due to difficulties getting RNA from hemolymph samples stored in RNAlater, Grace is testing out lyophilizing samples before extraction. Who knows what impact this will have on RNA, but it’s worth a shot!
Isolated RNA from three crab hemolymph samples preserved in RNAlater (Test 1, Test 2, Test 3) that had been lyophilized overnight last week.
Samples were provided by Grace.
I believe the primary purpose for this particular test was to verify that the freeze dryer was a feasible tool, since Grace experienced a minor mishap when she attempted the lyohpilization initially.
Lyophilization was successful, without any mess.
TEST 3 LYOPHILIZATION
Ran Bismark using our high performance computing (HPC) node, Mox, with two different bowtie2 settings:
- Default settings
- –score_min L,0,-0.6
The second setting is a bit less stringent than the default settings and should result in a higher percentage of reads mapping. However, not entirely sure what the actual implications will be (if any) for interpreting the resulting data.
Input data was previously trimmed per Bismark’s recommendation for Illumina TruSeq libraries (TrimGalore! 5′ 10bp):
List of input files and Bismark configurations can be seen in the SLURM scripts:
Gene Enrichment Results from
Mike fixed the gene enrichment tool, so I got to use it! Good thing, becuase ReVIGO is currently down and I cannot generate gene enrichment visualizations any other way. All of this information can be found in my R Markdown analysis notebook.
|The website expects inputs in the following format: sp
This means I don’t need to unfold the specific code from anything. The tool generates both reports and images, all of which can be found in this folder. I can also do comparisons between two different lists, which is useful. I used a p-value of 1e-2, the default, for each enrichment.
Figures 1-3. Gene enrichment visualizations for biological processes, cellular components, and molecular functions.
Exon and intron Uniprot codes
To get Uniprot codes for DML-Exon and DML-Intron overlaps, I used
intersectBed with the
-wao argument to find overlaps between these two files and the mRNA gff.
-wao allowed me to write out the overlaps between the DML-Exon or DML-Intron file with the mRNA and count the number of overlaps. If there was no overlap (which was unlikely), then the DML-Exon or DML-Intron file information was written out with NULL to indicate that particular exon or intron had no overlap in the mRNA file. I looked at the DML-Exon file in particular, and found it was really large. There seem to be a bunch of repeating lines. Not sure if that’s just because several genome chunks lead to different mRNAs, and thus, different exon patterns, or if there was a problem with the
-wao argument. This is something I will need to clean up after PCSGA.
Figures 4-6. Gene enrichment visualizations for biological processes, cellular components, and molecular functions.
Biological processes comparison
from the responsible grad student https://ift.tt/2Nlkghj