January 19

1/18/18

Group 1

  • Challenges
    • data varies greatly
    • time and cost constraints
    • used antibiotic surrogates to examine ecological health
  • Application
    • MOTU: molecular operational taxonomic unit
    • APDP: amplicon pyrosequencing denoising program
    • dbRDA
    • TITAN: threshold indicator taxa analysis

Group 2:

  • Purpose
    • method of metaborcoding used to morphology
  • Challenges
    • distinguishing active and inactive cells
    • interpretation of HTS
  • Application
    • extracellular DNA
    • distinguishing living and dead cells

Group 3

  • Purpose
    • distribution through habitat types
  • Challenges
    • comparisons limited to a low sample number
    • lack of standardization between studies
  • Application
    • distribution depends on habitat

Group 4

  • Abstract: geographic separation in two distinct lineages
  • Purpose
    • geographic separation causes genetic variation
  • chellenges
    • hard to genetically characterize organisms
  • application
    • cox – 1 gene might work four our purposes

Group 6

  • Purpose
    • effects of soil and vegitation on protists
  • Challeneges
    • lack of protist specific primers
    • most studies focus on DNA and not rRNA
  • Application
    • separately analyze ribosomal RNA and DNA

Group 7

  • Purpose
    • realize large diversity of soil parasites protists

Group 8

  • Purpose
    • wants to make universal Eukaryotic taxonomy database
  • challenges
    • no databases at the moment
  • Application
    • we could use UniEuk in our study

Isopycnic Centrifugation: Uses tiny glass particles to change the density of the centrifuge substrate

Category: benjamin_hamilton's notebook | LEAVE A COMMENT
January 19

1/11/18

Metabarcoding: the combination of DNA taxonomy and high-throughput sequencing, as a tool for the rapid assessment and monitoring of biodiversity in mixed, bulk samples

How To Clasify

  • Microscopic
    • difficult to isolate
    • many are morphological similar
  • Molecular
    • genomic diversity is unknown
    • standard methods are not established

3 Ways to Extract DNA

  • Single Isolate ID
  • Metabarcoding
  • Metagenomics
Category: benjamin_hamilton's notebook | LEAVE A COMMENT
October 24

Lab 10: Malarial Genomics

  • Purposes
    • Understand the Life cycle of P. falciparum
    • use the CDC and WHO websites to explore the facts about malaria and the strategy to reduce the global impact of the disease
  • Think Pair Share Exercises
    • Part 1: WHO Report
      1. 212 Million malaria cases in 2015
      2. 429,000 Malaria deaths
      3. malarial cases are falling
      4. Populations most susceptible to malaria are people living in the areas where transmission of the disease is highest- areas in Africa and the Saharan region. Children under the age of 5 have an under developed immunity against the disease, putting them at greatest risk for infection.
      5. The trends in Malarial intervention encompass vector control including insecticide nets or spraying insecticide inside of living areas.
      6. The best treatment are the ACT’s as they are a combination of numerous drugs
      7. Challenges to reducing malaria by 90% by 2030 involve funds to manufacture insecticides and the ACT’s, and dispersing those treatments to the needed populations. Advocating more governments to allocate more money to treating malaria. Understanding resistance to the drug and researching how we can combat the development of said resistance.
    • Part 2: Epidemiology and Challenges
      1. Additional host factors could include the education and awareness about Malaria, as people who are knowledgable of the disease are more likely to take preventative measures. Variability of genes and genetics of the host could affect infection as well.
      2. Agent/ Vector Factors: there are four different major specifies of Plasmodium that cause the disease, which creates issues for a drug that can combat all of these species, or to design a drug that can treat each individual species.
      3. Environmental Factors: Genetic variability in the area and population could be an environment that increases or decreases the presence of malaria.
    • Part 3: Systems Biology
      1. Certain natural features of regions can hugely affect malaria transmission as more water in areas serve as breeding grounds for the mosquitoes. Humidity and climate change can aid in mosquito reproduction as more habitats increase mosquito populations.  Economics also affect Malaria transmission as certain country’s governments can allocate more money to fund the treatment and prevention of malaria. Individual people also can advocate for their own health if they have the money to buy treatments including ACT’s and nets. Certain cultures may emphasize more natural/ homeopathic measures to treat Malaria which could be an incompetent way to fight the disease. Politics impact malaria infection as politicians can advocate for their people to receive treatment or educate their citizens about the disease and prevention.
      2. Are there certain initiatives encompassing global warming that could decrease populations of misquitos that thrive more in warm and wet conditions?
    • Part 4: The Life Cycle of P. Falciparum
    • Part 5: Genomics and Drug Resistant Malaria Treatment
      1. How feasible is it to distribute a malaria vaccine across African populations? What about the malaria vaccine/ ACT has made developing the treatment so hard to develop and distribute?
      2. The role genomics can have in addressing these questions involves how gene variability contributes to resistance of the drug in populations
      3. https://academic.oup.com/jid/article/185/3/380/896026/Molecular-Markers-for-Failure-of-Sulfadoxine
        1. What are the considerations and difficulties in developing an effective drug for Malaria?
        2. What are the genetic considerations in people’s resistance to such drugs?

 

Category: e_atwood's notebook, Uncategorized | LEAVE A COMMENT
September 26

Lab 6 (9/26/17)

  • Objectives/ Goals
    • Review Annotations for Stewie Griff, ensuring q_:s_ values from NCBI and PhagesDB are included
    • Become aquatinted with the future steps needed to publish the StewieGriff annotations
  • Procedure
    • Review Annotations; make sure all genes in your region are correctly annotated and entered in PhageNotes
    • Start to Annotate Phage Yeezus Genes #17-25
    • Go through the steps of final annotations and submit the completed project to University of Pitt for Quality Control
    • Submitting Completed Project
      • Make a final DNAMaster File that has all the final annotations in the Notes box.
      • Duplicate the Final File and make a file with just the functions in the notes box.
      •  Make an author’s list
      •  Make a cover sheet
  • Results
    • Gene #5 revised and Reviewed annotation 
      • Start: 800bp Stop: 2290bp FWD GAP: 7bp Overlap SD Final Value: SD Score: -3.146 (Best score) Z-Value: 2.897 CP: The gene is not covered The gene is slighly uncovered, but there are no other options SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: terminase [Arthrobacter phage Elkhorn], q:1 s:1 E-Value: 0.0 CDD: YmfN E-Value: 1.44e-11 PhagesDB BLAST: Lore_4, terminase large subunit, 496, q:1 s:1 E-Value: 0.0 HHPred: YmfN, Phage Terminase like-protein E-Value: 2.3e-34 LO: Yes ST: Agrees with Starterator F: terminase, large subunit FS: NCBI, HHPred Notes:
    • Gene #7 Revised and Reviewed Annotation 
      • Start: 2549bp Stop: 3709bp FWD GAP: 51bp Gap SD Final Value: SD Score: -4.629 (Best score) Z-Value: 2.16 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: portal protein [Arthrobacter phage Elkhorn] q1:s1 E-Value: 0.0 CDD: Phage_portal E-Value: 1.73e-18 PhagesDB BLAST: Lore_6, portal protein, 386 q1:s1 E-Value: 0.0 HHPred: HK97 Family Phage Portal Protein; Phage, HK97 family, Portal, Corynebacterium; HET: GOL, SO4, PO4; 2.9A {Corynebacterium diphtheriae} E-Value: 1e-33 LO: ST: Agrees with Starterator F: portal protein FS: NCBI Notes:
    • Genes #17-25 to be Annotated for Phage Yeezus
    • Gene #17:  SSC: Start 8628 Stop 10130 GAP: 4 CP: SD: -1.767 Z: 3.034 SCS: Agree NCBI Blast:terminase large subunit [Arthrobacter phage Amigo] PDB BLAST: SorJuana_22, terminase, large subunit, 500 
      HHPred:Gene 2 protein; DNA packaging, terminase, ATPase, nuclease; 1.69A {Shigella phage Sf6} e-value:4.2e-33 LO: yes ST: F: FS:
  • Future Work
    • Continue Annotating Genes #17-25
    • Work on Publishing Stewie griff Annotations
  • Notes
    • E-Value: for HHPred, if the e-value is not close to zero, just record “No good hit”
    • https://discover.kbrinsgd.org/autoannotate/
    • on DNA master, ideal Z-value greater than 2.0
    • SD Score- the least negative, the better the score
    • NCBI — protein blast — enter the product from the saved dna master file, scroll down to query and sequence scores (q#:s#)
    • higher z score, lower final score (closet to zero), longest # ORF
    • HH Pred
      • hhPRED Search in google, search, HHpred, on DNA master: sequence, feature drop down to gene #, right click, copy as translation
      • make sure COG, PDB, p pham are selected
    • LO: longest open reading frame (yes/ no)
Category: e_atwood's notebook | LEAVE A COMMENT
September 15

Link Annotations 9_12_17

  1. Link Gene 10
    • SSC: Start: 6014 Stop: 6436 GAP: 5 CP: Covered SD: -3.763 Z: 2.597 SCS: GeneMark and Glimmer agree NCBI Blast: major tail protein [Arthrobacter phage Decurro] E-value: 5e-95 PDB BLAST: Yank_10, major tail E-value: 1e-76 LO: Yes F: NKF FS: NKF
  2. Link Gene 11
    • SSC: Start: 6436 Stop: 6813 GAP: 1 CP: Covered SD: -7.726 Z: 1.202 SCS: GeneMark and Glimmer agree NCBI Blast: minor tail protein [Arthrobacter phage Decurro] E-value: 1e-84 PDB BLAST: Yank_11, minor tail E-value: 1e-67 LO: Yes F: NKF FS: NKF
  3. Link Gene 12
    • SSC: Start: 6820 Stop: 7170 GAP: 6 CP: SD: -3.016 Z: 3.350 SCS: GeneMark and Glimmer agree NCBI Blast: hypothetical protein SEA_DECURRO_12 [Arthrobacter phage Decurro] E-value: 4e-79 PDB BLAST: Yank_12, function unknown E-value: 9e-62 LO: Yes F: NKF FS: NKF
  4. Link Gene 13
    • SSC: Start: 7281 Stop: 9278 GAP: 110 CP: Covered SD: -4.389 Z: 2.214 SCS: GeneMark and Glimmer agree NCBI Blast: tape measure protein [Arthrobacter phage Decurro] E-value: 0.0 PDB BLAST: Yank_13, tape measure E-value: 0.0 LO: Yes F: Phage-related protein E-value: 2.06e-10 FS: CDD
  5. Link Gene 14
    • SSC: Start: 9272 Stop: 11089 GAP: 7 CP: Covered SD: -2.815 Z: 3.029 SCS: GeneMark and Glimmer agree NCBI Blast: minor tail protein [Arthrobacter phage Decurro] E-value: 0.0 PDB BLAST: Decurro_14, minor tail E-value: 0.0 LO: Yes F: NKF FS: NKF
  6. Link Gene 15
    • SSC: Start: 11092 Stop: 11607 GAP: 2 CP: SD: -2.909 Z: 2.991 SCS: GeneMark and Glimmer agree NCBI Blast: hypothetical protein SEA_JESSICA_16 [Arthrobacter phage Jessica] E-value: 6e-121 PDB BLAST: Yank_15, function unknown E-value:1e-93 LO: Yes F: NKF FS: NKF
Category: claire_costanza's notebook, Uncategorized | LEAVE A COMMENT
September 12

Phage Annotation #3 09_12_17

Background: We have currently been working through the process of annotation and understanding the different tools/programs involved in our annotation process. We have annotated Link Gene 2 in class in order to reinforce this process.

Purpose: Master the ability to annotate the structural features of a predicted gene. Master the knowledge of the meanings for each shortcut in the annotation template. Explore the use of Phamerator to discover whether a gene function is conserved and compare start sites of published genomes. Explore the mosaic nature of phage genomes using Phamerator and analyze the differences between closely related genomes. Use BLASTp and the CDD to explore preciously predicted gene function.

Procedure:

  1. Annotation Review and Practice (save in notebook/blog for later use)
    • Open your saved auto-annotated Link DNAMaster file
    • Check your DNAMaster settings
    • Open the Guiding Principles and use to assist in annotation (remember to use the template SSC: GAP: CP: SD: Z: SCS: NCBI Blast: PDB BLAST: HHPred: LO: ST: F: FS:)
      • Use GeneMark for CP, NCBI for NCNI Blast, and phagesdb for PDB
    • Annotate Gene 9 on your own
      • Find the most likely start codon instead of the default start codon setting
  2. Introduction to Phamerator
    • Practice using Phamerator with annotated gene
  3. Introduction to CDD (Conserved Domain Database)
  4. Annotation practice

Results:

Conclusion:

In lab we have fine tuned our annotation process for most of the template that we are using.

  1. Link Gene 3
    • SSC: Start: 591 Stop: 806 GAP: 30 CP: Covered SD: -6.444 Z: 1.190 SCS: No, we chose the longest ORF NCBI Blast: hypothetical protein hypothetical protein SEA_DECURRO_3 [Arthrobacter phage Decurro] E-value: 2e-46 PDB BLAST: TymAbreu_3, function unknown E-value: 9e-41 LO: Yes
  2. Link Gene 9
    • SSC: Start: 5652 Stop: 6008 GAP: 4 CP: Covered SD: -4.265 Z: 2.728 SCS: Do not agree Chose GeneMark NCBI Blast: hypothetical protein SEA_DECURRO_9 [Arthrobacter phage Decurro] E-value: 1e-77 PDB BLAST: Yank_9, function unknown E-value: 9e-63 LO: No

Future Work: Use the techniques and programs that we have become more familiar with to continue our progress with annotating genes.

 

Category: claire_costanza's notebook, Uncategorized | LEAVE A COMMENT
September 12

Phage Annotation 8/29/2017

Objective:

 

Our main objective is to create gene annotations by computational means. We will be identifying relative location of genes, protein encoding, and RNA encoding genes. Keeping in mind that these are all predictions but still authentic research.

Procedure:

  • Set Key Preferences
  • Importing a DNA Sequence
  • Auto-Annotating a Genome
  • Compare potential start and stop codons

Results:

We were able to successfully import a DNA sequence from the Amigo Download into the DNA Master software and Auto- Annotate the genome. We observed the start and stop codons for gene 24.

Conclusion/Future:

DNA Master is a tool that we will continue to use throughout the semester to further inspect and annotate genomes. In future labs we will analyze other aspects of genes and genomes.

 

SSC: GAP:CP:SD:Z:SCS: NCBI Blast: PDB BLAST: HHPred: LO: ST: F: FS:

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September 5

Auto-Annotation 9/5/2017

Purpose:

In this lab we will begin auto-annotating the Link.fasta file using PECAAN. Using GeneMark.hmm prokaryotic, we will be able to predict the coding potential for genes. We will also be comparing new sequences with sequences of  the known or assumed using BLAST.

Procedure:

DNAMaster Stetup

  • Open link in FASTA file
  • Go to https://discover.kbrinsgd.org/autoannotate/ and upload Linkfasta A file and enter phage name
  • Click “Process”
  •  Select and Copy the entire contents of the text box
  • click “documentation” on DNA Master and paste information
  • Parse information with default settings
  • Save genome to downloads

GeneMark

BLAST

  • Go to https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • select gene #2 from DNAMaster and copy and paste the information from the “product” tab into the space provided
  •  click BLAST and select the closest phage match to our gene
  • copy the name of the phage and put this in the “notes” of our DNAMaster

Auto-Annotation

  • Copy the template saved on DNAMaster from the previous lab into the notes section
  • Fill in template based on  information given on ORFS and RBS of gene #2

Results

Raw SD score: -2.839

Generic Z Value:- 2.641

Final Score:-4.441

Start Codon:345

Stop Codon:560

There was a 4bp overlap

 

 

 

Conclusions

We found  a solid match to our gene through the NCBI protien BLAST database named Anthrobacter_aurescnens_TC1.  The final score on the Annotation was -4.441, which is good because it is the closest to zero.

Category: jailyn_foster's notebook, Uncategorized | LEAVE A COMMENT
September 5

Lab 2: Gene Annotation

Title: Gene Annotation

Date: 9/5/17

Objective:

Our purpose is to learn how to prepare for auto-annotate on BLAST and Gene Mark.

Procedure:

  • download Link.fasta file from phages DB and add it to DNA Master
  • Go to https://discover.kbrinsgd.org/autoannotate/
  • select and copy the entire content on to Pecaan
  • Set Translation to Bacteria and Plant Plastid Code
  •  For BLAST, click on gene 2
  • click DNA, ORFS
  • click on start codon and click RBS
  • Go to DNA Master (product) and copy it to BLAST
  • For Gene Mark, Go to Gene Mark link on canvas
  • upload the Link.fasta file
  • use PDF and receive a document for the Gene Mark reading
  • For annotating in DNA Master, and type in notes

Results:

SSC: GAP:216 CP: SD: Z:-4.441 SCS: NCBI Blast: PDB BLAST:
HHPred:Agree E value: 4e-43 LO: Agree ST: F: FS:
Start Codon: 345
Conclusion:
The Link phage undergone many testings in bacteriophage databases such as Gene Mark and BLAST and both agreed to have the same name. Each database specializes in different functions. BLAST compares new sequences with sequences of known or assumed function. Gene Mark illustrated the gene strands to see if the gene was covered by the data analysis.
Future Plans:
I hope to be able to have my DNA Master to work on my own laptop so that I can easily store my information. Also, I hope to become familiar with all of the databases.
Category: ashley_washington's notebook, Uncategorized | LEAVE A COMMENT
September 5

Annotating Link Phage

5 September 2017

Title

Hypothesis or Goal:

Autoannotate a gene via the PECAAN Auto Annotation Tool as a workaround

Background:

Because databases no longer work with DNA Master Autoannotation, a workaround method is necessary to annotate genes. Now we are autoannotating through PECAAN.

Procedures:

Opened Link FASTA in DNA Master Open>FASTA Multiple Sequence File> Export > This sequence only

Performed autoannotation with work around method discover. kbrinsgd.org/autoannotate/ > Choose FASTA file> process>Paste result into documentation tab> Parse > Parse

Saved file to DNA Master folder

Make sure Translation Table is set to “Bacteria and Plant Plastid Code”

Open GeneMark and Run FASTA File

Begin Annotating using template

Run ORF – DNA> ORF> RBS

Run BLAST by Copying segment from Product tab on DNA Master and pasting to Blast on NCBI and Phagesdb

Observations:

GeneMark shows the coding potential for a gene

Coding Potential –The likelihood that a gene is located at a certain genome

BLAST compares the proteins from a phage to other phage proteins to try and identify the protein

Raw experimental data:

Data analysis:

Ideas for future experiments and next steps

Practice more gene annotations

Category: stephanie_sullivan's notebook | LEAVE A COMMENT