Gene in House Spiders
GENOMICS LABORATORY – FINAL SCIENTIFIC REPORT and ORAL PRESENTATION
This document provides an overview of the expectations for the final lab report and the final oral presentation for Genomics Lab (see syllabus). These two assignments are each worth 15% of your final grade for the lab course, so 30% in total. Your assignment is to synthesize the information you and your partner have learned about your gene of choice from the Parasteatoda tepidariorum (Ptep) genome. We are excited to learn about your interesting discoveries and for you to share them with the rest of the class. The paper and presentation will mirror a typical scientific paper and oral presentation as detailed below.
Work (Labs) we performed together for this project:
- Sequence editing and BLAST analyses of Ptep venom gland ESTs (Week 4)
- RNA-Seq differential expression analysis of transcripts in venom glands vs. non-venom tissues (Week 7)
- Exploring gene of choice, its structure and genomic neighborhood (Week 8)
- (upcoming) Mass spectrometry analysis of house spider venom (Next week)
Plus, Original/Independent Research you and your partner performed together for this project once you chose your gene.
Tools available to you include the following: NCBI, BLAST, MEGA (alignment and phylogenetic trees), DNA Subway, RNA-Seq Ptep 8 library data files, NCBI and i5k Parasteatoda tepidariorum genome browser and BLAST tools, EMBOSS and ExPASY tools – it’s up to you!!! Below we list some possible questions you can explore with these tools (see Methods section “Hints and suggestions of avenues to explore in your research” for some ideas that will lead you along the path to discovery). At the end of the document we provide some links to useful resources.
SECTIONS OF THE WRITTEN LAB REPORT (in the form of a scientific paper)
I. Title:
Come up with a title for your lab report that cohesively describes the focus of your project.
II. Abstract:
Summarize your report including a brief description of the background, purpose, methods and results. Remember that the abstract should provide a snapshot of your entire work in no more than 300 words.
III. Introduction
This section should provide the context and background information for your study. Provide some detail on the organism we dealt with and the genome of this organism. Provide some details about your gene of choice and what was previously known about it, if anything. Why is your study important? Provide any additional background necessary for understanding your study. What were your major objectives? If you have managed to develop a hypothesis be sure to explain what the hypothesis is and how you plan to test or investigate the hypothesis. Alternatively you may propose a question that the study seeks to answer.
IV. Methods
Provide a brief description of the methods we used in the labs performed together, as well of description of additional methods you used to learn new information about your gene of choice and its context in the genome (We provide hints and suggestions of avenues to explore next). You are not required to specify very extensive details of the methods used. Just provide enough information that would enable others to replicate your study, do mention the names of specific software used, and names of any analyses.
Hints and suggestions of avenues to explore in your research
So you have a gene on a genomic scaffold from the house spider genome – now what?? This is where you have license to flex your intellectual muscles and think creatively about how to harness the power of some of the tools you’ve learned about this semester [Tools available to you: NCBI, BLAST, MEGA (alignment and phylogenetic trees), DNA Subway, NCBI and i5k Parasteatoda tepidariorum genome browser, EMBOSS and ExPASY tools, RNA-Seq data, Ptep transcriptome fasta file – it’s up to you!!!]
Next – some possible questions – We are not saying you must explore all of these questions, or any of these, they are suggestions you are welcome to explore or ignore in favor of other ideas. For each question, think about what method or tools you would use to find out the answer….ask someone how to approach the question if you cannot figure out how.
- What is the function of this gene and the protein it encodes? If there are no BLAST hits, does it have GO Terms of protein domains that might hint at its function? What does the RNA-Seq expression data say about possible function?
- What is the structure of this gene (i.e. map number and relative position of introns, exons, UTRs etc.; Does my gene produce one or more proteins, do they have similar expression patterns?
- What other genes surround my gene of choice on its genomic scaffold? Are they related to my gene of choice evolutionarily?
- Does my gene have orthologs or homologs in other species?
- Is my gene part of a gene family in its genome? If so – how big is this gene family, where are the other members of the family located in the genome (are they on the same or different scaffolds?), how similar are the different member of the gene family? Do they seem to have different functions?
- V. Results
Describe the major findings of your study – what did you learn about your gene of choice, its structure and its place/context, function or evolution in the genome and in relation to other genes within this genome and in other organisms (remember: your question are up to you). You can use figures and tables to show your results, but you should also include the results in the form of text that allows readers to understand your major results and any figures. Any figures and tables should have legends and be referred to in the text of the results section.
- VI. Discussion and Conclusion
Discuss the results you obtained. What results did you find were novel?, which are important?, why are they important? which deserve to be followed with additional studies and what studies should be pursued in the future given what is still unknown? How should those studies be designed?
- VII. References
Include any references you used to write your report. We expect to see a few references.
Parasteatoda tepidariorum Alpha-latrocrustotoxin-Lt1a-like (LOC107440043) Gene in House Spiders
Student’s Name
Affiliation
Course
Professor
Due Date
II.Abstract
The increasing sophistication of sequencing methods provides an opportunity for biomedicine to understand the genome of various eukaryotes with important applications in medicine. The study of venom has been instrumental in the development of medicine and antitoxins. With next-generational sequencing, there is an opportunity to expand the existing knowledge on venoms. The study of Parasteatoda tepidariorum alpha-latrocrustotoxin-Lt1a-like (LOC107440043) provides an opportunity to understand the evolutionary transformation of venom protein composition. The gene is responsible for the production of venom proteins in house spiders (Parasteatoda tepidariorum). Though the house spider shares the same family as the black widow, its bite induces less severe pain in humans compared with that of the black spider. An understanding of the venom gene produced in the house spider and its differentiation with the venom protein from the black widow can yield important insight that can contribute to an increased understanding of venom. The purpose of the current study is to add to existing knowledge by studying the genomic structure of the Parasteatoda tepidariorum alpha-latrocrustotoxin-Lt1a-like (LOC107440043) gene and describe some of the proteins that are transcribed by the gene. To this end, the researchers take advantage of the advancement in sequencing and rely on BLAST to identify the protein produced by the gene. Notably, the gene does not produce venom with similar toxicity levels as the one produced by the alpha-latrocrustotoxin gene in the black widow.
III.Introduction
The biomedical application of animal venom as well as its utility in modeling the origin and diversification of genes in species leads to raised interest in the study of animal venoms. Animal venoms are largely made up of proteins that have been adequately studied by scientists. Nonetheless, there has been minimal focus on the study of most venomous species leading to minimal understanding of the evolution of dangerous toxins (Gendreau et al., 2017). Nonetheless, there has been rapid development in the availability of next-generation sequencing (NGS) technologies that have focused on describing the whole genomes of eukaryotes considered essential. The i5K initiative that has managed to sequence the house spider (Parasteatoda tepidariorum) is one of the efforts established to study important eukaryotes (i5K Consortium, 2013). House spider shares a similar family as the black widow, but its venom is far less venomous.
The venom produced by the black widow is famed for its potency, with the α-latrotoxin gene considered a contributing factor to the potency of the venom. α-latrotoxin is the only latrotoxin gene recorded as having toxic effects on vertebrates. Advances in NGS led to the discovery of more than 20 unique latrotoxins that are expressed within the venom glands of the black widow (He et al., 2013; Wang et al., 2022). Given the absence of equally toxic proteins in house spiders, Haney et al. (2019) have noted that the study of the genome of the house spider will shed light on the alternative transcription of venom proteins. In the present research, a sample of nucleotide sequence from the venom glands of the house spider is sequenced and transcribed.
IV.Methods
Preparation and Nucleotide Sequence Translation using EMBOSS. The sample was put in RNA sequencing tools to assess the quality of the raw data and facilitate accurate results. The edit application group of EMBOSS was used to clean the raw data by trimming the bad quality region. Once the quality was addressed, EMBOSS was used to translate the nucleotide sequence to a protein sequence that could be identified.
BLASTx. Once the protein query was generated, BLASTx was used to identify the protein transcripted by the nucleotide sequence. The file containing the query was uploaded to the program, it was searched in the protein database and the resulting protein was identified.
BLASTn and NCBI database search. The nucleotide sequence was subjected to a BLASTn search, and the results in which the fourth column “species hit” was to P. tepidariorum and if the third column accession number started with the prefix XM, NM, or XR were recorded in a table. The NCBI database was used to identify the gene to which the nucleotide sequence BLAST. In addition, the q-value of the transcript and the fold changes were retrieved from the NCBI database. The NCBI database was also used to identify other genes in the same genomic scaffold and return the complete gene sequence of the nucleotide sequence.
Protein Identification using Mass Spectrometry: Mass spectrometry was used to identify the expressed protein from the sample of the nucleotide sequence.
V.Results
The sample had 24570956 sequences, with a length range of 20-100. While the overall quality of the sequence was good, the quality declined as the length of the sequence increased as evidenced by the reduction in the total number of sequences to 24569923 following the removal of poor-quality reads. The longest predicted transcript from the sample was XM_043041225.1, with a length of 61527.7. A BLASTx analysis of the XM_043041225.1 showed that it was a protein named titin (Parasteatoda tepidariorum).
BLASTn analysis of the sample returned eight results that were either ‘species hit’ to P. tepidariorum or had the prefix NM, XM, or SR as shown in table 1 below. The ascension number from BLASTn analysis returned the accession number XM_016052822.2 and it BLAST to delta-latroinsectotoxin-Lt1a-like [Parasteatoda tepidariorum]. The q-value of the transcript was 0.15198 (table 2), which is larger than the p-value. The inability of the expression to meet the critical value even though it is heavily expressed in venom tissues could be attributed to the addition of non-venom glands in the calculations. Silk tissue type had the largest divergence in expression, while ovary and venom appeared the most dissimilar to each other in their expression across transcripts.
TPM values
Blast hit
(copy column 2 above)
Accession #
(copy column 3 above)
Venom1
Venom2
Silk1
Silk2
Ovary1
Ovary2
Ceph1
Ceph2
aldehyde dehydrogenase, dimeric NADP-preferring-like [Parasteatoda tepidariorum]
XR_006226408.1
0.501836
2.92E-05
6.00047
0.0440886
16.2086
21.3155
5.62312
6.73966
Parasteatoda tepidariorum actin-interacting protein 1 (LOC107454932), mRNA
XM_043051009.1
25.5519
23.9068
25.3058
19.3426
121.88
134.77
27.4716
24.7545
Parasteatoda tepidariorum very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (LOC107452655)
XM_016069201
143.537
128.643
5.0452
2.55923
19.5184
21.5985
3.26254
4.79559
Parasteatoda tepidariorum very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (LOC107452655)
XM_016069201
143.537
128.643
5.0452
2.55923
19.5184
21.5985
3.26254
4.79559
Parasteatoda tepidariorum alpha-latrocrustotoxin-Lt1a-like (LOC107440043)
XM_016052819.3
6075.25
6619.78
0
0
46.4078
41.2751
40.8637
40.293
Parasteatoda tepidariorum delta-latroinsectotoxin-Lt1a-like
XM_016052822.2
4064.08
5326.1
0
0
32.6076
31.0206
30.433
28.7507
Parasteatoda tepidariorum uncharacterized LOC107442349
XM_016055901.2
25356.7
26536.6
0
0
198.068
225.304
195.095
185.506
Parasteatoda tepidariorum uncharacterized LOC107454127
XM_016071197.2
5.35902
11.2341
50.0044
62.2089
3.98946
3.78224
242.004
482.147
Table SEQ Table \* ARABIC 1: BLASTn Results
Of the identified results, XM_016052819.3 (Parasteatoda tepidariorum alpha-latrocrustotoxin-Lt1a-like (LOC107440043), mRNA is on scaffold ptep_3.0 scaffold 75 with the full length of the scaffold being 1892500 bp. The coordinates of the gene on the scaffold are 1,287,266 - 1,293,397. The gene has 1 exon and no introns. In addition, the gene does not have an alternative transcript and part of the XM_016052819.3 covered by the EST is P8-A12_G12_084_2017-10-18. The TPM values of the gene are indicated in Table 1 above. The other example of the gene that occupies a similar genomic scaffold as XM_016052819.3 are mRNA-alpha-latrocrustotoxin-Lt1a-like, mRNA-alpha-latroinsectotoxin-Lt1a-like, mRNA-delta-latroinsectotoxin-Lt1a-like, and mRNA-double-stranded RNA-binding protein Staufen homolog 2. The complete gene sequence of Parasteatoda tepidariorum alpha-latrocrustotoxin-Lt1a-like (LOC107440043), mRNA is detailed in Appendix 1.
Blast hit
Accession #
q-value
B(fold-change)
aldehyde dehydrogenase, dimeric NADP-preferring-like [Parasteatoda tepidariorum]
XR_006226408.1
0.37037
-3.3907
Parasteatoda tepidariorum actin-interacting protein 1 (LOC107454932), mRNA
XM_043051009.1
0.98302
0.15222
Parasteatoda tepidariorum very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (LOC107452655)
XM_016069201
2.7086e-15
3.662
Parasteatoda tepidariorum very-long-chain (3R)-3-hyd...