BlueSlugg

COMMON ERRORS

• Make sure you are not putting a nucleotide sequence into a protein query, and vice-versa.

GENSCAN

http://genes.mit.edu/GENSCAN.html

Intr - internal

Term - terminal

Prom - promoter

Sngl - single

FegenesH, Animal Version - Ab initio gene prediction (SOFTBERRY)

http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind

GeneID

GeneFinder

Notes

Chimeric Sequences

t(9,22)(q34,q11)  Chromosomes 9 and 22 are affected. Locations q34 and q11 are affected respectively.

Comparison of Fgenesh (softberry) and Genscan.

(MUST BE IN FASTA FORMAT)

NG_011759 both return 15 exons total with 11 on one stand, and 4 on the other.

EU445484 Genscan - 7 exons. Softberry - 6 exons.

AF083883 Both returned a result of 3 total exons.

Y16787 GenScan returned a result of 6 exons, while Fgenesh returned a result of 8 exons. In reality there are 7 exons total. Fgenesh (Softberry) had results that were superior to GenScan.

Notes from prof and students

Use both in the lab practical. BLAST putative exons for quality control. GenScan uually had the exons in the right location. Fgen was generally wrong with this regard. Fgen was more sensitive  one out of the 4 cases.

Bacterial Genomes

When accessing a particular region at NCBI search for the ID number of the gene, then click on “change region shown”.

fgenesB

• don’t forget to select the given organism

http://www.softberry.com/berry.phtml?topic=fgenesb&group=programs&subgroup=gfindb

Notes: Tu/Op = transcription units/ operons

Glimmer

http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi

GeneMark (preferred method)

• Note, GeneMark not only creates a prediction, but gives alternative predictions and their likelihood.

http://www.ncbi.nlm.nih.gov/genomes/MICROBES/genemark.cgi

MRSA sequence NC_002952 GeneMark accurately predicted two of the three genes, which was closer than either of the two other two other programs. It is agreed in  class that GeneMark is the superior overall program.

NC_012578 GeneMark and Softberry both analyzed the sequence and predicted accurately, however Softberry requires the selection of a particular organisms -_-

NC_002505 - GeneMarkined up the numbers perfectly! Softberry was close.

Secondary Structure Prediction (for proteins)

alpha helix

beta strand

coils, turn disorganized region

http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml

https://predictprotein.org/

http://cib.cf.ocha.ac.jp/bitool/MIX/

http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=6043

With regards to CD4, all three structural prediction proteins on the Japanese website turned out to be utterly useless. All three programs predicted an abundance of alpha helices, yet the crystalized structure contained none. I feel like I am spinning chicken bones to try and predict the harvest with these useless programs. (Note, when I used these programs during my practice practical for a general bioinformatics class, these tools turned out to be more useful.)

predictprotein.org is far superior to the three rotein prediction methods on the Japanese website.

Use cn3d to view the 3D protein structure. Don’t forget Style - Coloring Shortcuts - econdary Structure

Protein Location Prediction Tools

PSORT II - Protein Sorting

http://psort.hgc.jp/form2.html

PROTCOMP

Protein Compartment

http://www.softberry.com/berry.phtml?topic=index&group=programs&subgroup=proloc

Between Protcomp and PSORTII, Protcomp accurately predicted the location of CD4 (plasma membrane) however PSORT II did not.

Protcomp accurately predicted the location of all 3 isoforms of CD4. It said that all three were located in the plasma membrane.

Note: Protcomp was inaccurate for NP_000509 at first, however when a FASTA format was copy/pasted into the query, instead of the version of the sequence at the bottom of the protein database that includes numbered lines etc., the protein prediction came back accurate.

Conclusion for Protein Prediction Location

Both programs have similar degrees of accuracy. Use both programs to predict protein locations and compare the predictions.

Discrepancy During Lab Practical

Make note of the respective predictions for each program. Rationally interpret the results. One prediction may need to be discarded, or it might have a close tie between two predictions. Also remember that protein locations are not static within the cell, proteins move from one location to another, and can often be found in multiple locations.

In Professional Setting

Follow the steps during the Lab Practical discrepancy, as described above. Additionally, it may be a good idea to search the professional databases (such as NCBI) and infer the location of this protein based on similar proteins.

Transmembrane Proteins Prediction

(When comparing to NCBI ctrl F for “transmembrane region”)

Sosui - (may require firefox.)

In Windows go to the control panel. May need to allow the Sosui website, and update Java.

In Mac Click the security tab in FireFox, edit site list, add Sosui.

http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html

TM Predict

http://www.ch.embnet.org/software/TMPRED_form.html

TMHMM2

http://cbs.dtu.dk/services/TMHMM/

Helical Wheel program - A great compliment to the other three programs, a section of a protein sequence thought to be a helical structure can be pasted into this program for analysis. (NOTE: This program assumes a-priori that any sequence caopy/pasted into the query box is a helical region.)

http://rzlab.ucr.edu/scripts/wheel/wheel.cgi

Main site for the Helical Wheel lab lab

http://rzlab.ucr.edu/

Class verdict - all three programs were pretty accurate and performed with minimal error.

Protein Structural Prediction

Predicts signal to go to the ER and associated cleavage site:

Signal P

http://www.cbs.dtu.dk/services/SignalP/

Signal P explaination

http://www.cbs.dtu.dk/services/SignalP-4.1/output.php

Disulfide Bonds (Cysteine - Cysteine bonds) (See also the Prosite Tool under Protein Signatures)

CYS_REC

• Used to predict the presence of disulfide bonds in a protein.

http://www.softberry.com/berry.phtml?topic=cys_rec&group=programs&subgroup=propt

Predict coiled-coils

COILS

http://www.ch.embnet.org/software/COILS_form.html

Marcoil

http://toolkit.tuebingen.mpg.de/marcoil

COILS/ PCOILS (not used in BAM class)

• Predicts coiled coils and compares the prediction to a database of known sequances.

http://toolkit.tuebingen.mpg.de/pcoils

Repetitive Elements Search

Nucleotide Blast for dbALU database. Finds alu repetitive elements in your nucleotide sequence query.

http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

Leucine Zipper

2zip Server

http://2zip.molgen.mpg.de/

Protein Signatures

Scan Prosite Tool - Tool excludes motifs with high probability by default (this is a good thing).

http://prosite.expasy.org/scanprosite/

Fingerprint Scan - A tool that scans for motif fingerprints.

http://www.bioinf.manchester.ac.uk/cgi-bin/dbbrowser/fingerPRINTScan/FPScan_fam.cgi

Nucleotide Signatures

Search for Human Promoters (nucleic acid analysis) - A threshhold of .8 is used by default to avoid false positives (this is a good thing).

http://linux1.softberry.com/berry.phtml?topic=fprom&group=programs&subgroup=promoter

CpG - Searches for CpG islands.

http://www.softberry.com/berry.phtml?topic=cpgfinder&group=programs&subgroup=promoter

Searching with Putative Chimeric Proteins

BLAST-P Against rough-seq database

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

Miscilaneous Bioinformatics Links

http://cbs.dtu.dk/services/

http://cbs.dtu.dk/biolinks/

3D Visualization and Prediction

CN3D

Download http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3dwin.shtml

CN3D Exercise

α, β, α/β, α+β, and a transmembrane protein