Bioinformatics and BioStatistics Method Forum

Determining the N- and C-terminal regions in a protein

Fri, 10 Feb 2012 09:48:01 +0000

Dear all,

So, something's been bugging me all night. I'm doing a literature review on the polyester synthase and a group of related proteins. Coming to the molecular aspect, I encounter the terms "N-terminal / C-terminal regions" often.

How do we (or in the near future, I) determine / decide where the N-terminal or C-terminal spans?

The first thought that came into my mind was a multiple alignment with the same protein in other organisms. But that’s only useful for those having a conserved N or C regions. What more, this particular group of protein has a “non-conserved N-terminal region” while the alpha/beta hydrolase fold domain is conserved in part of the C-terminal (see below).

1......98.............................................589
NNNNNCCCCCCCCCCa/bCCCCCCC

The regions based on the protein of the model organism. (conserved region in blue)

Until now, only the protein in the model organism is well-studied and annotated. So, another way is to align the amino acid sequence from the model together with those from other organisms. Where the C-terminal begins (aspartate 99), that position will be a benchmark of sort to the other query sequence. Still, I felt this isn’t very convincing.

Waitiing for some feedbacks / suggestions / pointers regarding this.

Thank you

BLASTing protein block

Sun, 29 Jan 2012 13:32:52 +0000

Hi
I have a question, I generated protein block, which represent a conserved domain (moti) in a family of proteins I'm working on, now I would like to BLAST this motif, How can it be done ?

thanks
Ofir

how to find copy number for different alu subfamilies

Thu, 26 Jan 2012 23:05:35 +0000

Is there a easy way to find out the copy number of invidual alu subfamilies in some database ?? Its possible to identify the subfamilies in repbase but i find it really tricky to get a full report over the copy numbers =/

Please help me=)

Determining gene of interest from Cell culture lines

Wed, 25 Jan 2012 02:29:34 +0000

Hi there, I am a summer research student currently performing PCRs to check for the presence of opioid receptors in various cell lines.

What I've done so far is to extract mRNA from cell cultures (e.g. 4T1, Raw 264.7 and H5V), doing a reverse transcription and performing PCRs on the cDNA to check if there are any opioid receptors present.

My PCRs have been very inconclusive ( I usually get primer dimers) so I'm trying to check and see if my primers will actually work on the cell lines.

However! BLAST is extremely confusing and I have no idea if it's possible to check my primer design against cell lines. Where can I get the genomic sequence for 4T1, H5v etc, etc.

Please, any enlightenment on the topic will be most appreciated!

~Byong

Timeline statistics

Mon, 16 Jan 2012 13:49:34 +0000

Hi Everyone, I am a PhD student with some difficulties about what test to choose to verify an experiment. Hope you can give me some help!

My experiment:
I am counting the number of a subset of cells in Drosophila overtime.
I have a mutant and a wild type control. I dissect the flies at different time points: just eclosed, 1 day after eclosion, 3 dae, 6 dae, 9 dae, 12 dae and 15 dae. For each time-point I have a n number of around 50.

The problem:
What happen with these cells is that in the wild type population the number of cells does not decrease overtime, while in the mutants it does. Moreover the wild type population has an average of 6 (in all the dissection days), while the mutant starts with 4 and goes down to 2. Maybe if they were the same amount initially I could have used an anova test with a posthoc test (like tukey) to show the difference between wild type and control for each day, but in this case an anova would tell me only that all the wild type are different from the mutants (even at eclosion!) because of the basic difference they have, or that for example mutant dae15 is different from mutant dae0,1,3. Moreover, what I would like to test against is not that the wild type is different from the mutant at a particular timepoint, but that the mutant numbers decrease faster than the control numbers.

Possible solutions:
I was thinking that one possibility would be to do an anova on a normalized sample: I could divide every number (and I mean the single numbers, not the average) I get for the average of the eclosion day, and do an anova on that ratio (So I would have liked normalized the two populations to the eclosion day and it would not matter anymore that their raw number are different), but something tells me I would mess up all the statistics: I am not sure if this approach is statistically correct.

Another possibility I was thinking is to use some kind of correlation or regression analysis, but in the example I found I never found this approach on my kind of experiment, so i am not sure how to work this out. In the various handbook it always show this kind of approach to smaller experiments, like comparing blood pressure and coffee assumption, and with much less numbers.

If you have any idea how this problem could be solved, I would be grateful!

Searching genome with specifed number of Ns between two known sequences

Mon, 16 Jan 2012 09:37:05 +0000

Do you know if there's a way to search a genome in BLAST for sequences separated by, say, 5-30 N? E.g. AAAA (N)_5-30 TTTT?

Probability of E.coli 2 mutations that lead to a lysine residue.

Sat, 14 Jan 2012 17:25:02 +0000

Just doing a reality check to see if my number is right. What would the probability of a single lysine residue arising from an alanine (has to be a two base change) with a mutation rate per base per replication of 5E-10.

Thanks Much

Need help with basic bioinformatics

Thu, 12 Jan 2012 02:11:42 +0000

Hey,

Just a little background on myself, I'm a new grad student in a microbiology lab. I have begun to realize that bioinformatics is really important and I admit I'm atrocious in this area. I want a sort of starting point to better my knowledge. Mainly I want to be able to look up DNA and protein sequences, and also make comparisons between two sequences (DNA or protein) which I have looked up. Sadly I don't even know how to do this properly. If anyone can give me some step by step info on how to do this, it would be great.

Thanks

curve fitting - growth modelling

Wed, 11 Jan 2012 16:05:15 +0000

hi guys,

I'm just wondering, has any of you done any statistical modeling of plant growth? I know, plants normally have a s-shape growth pattern, but under certain conditions (e.g. disease, drought) they don't follow this kind of growth curves.

My data shows more of a parabolic curve, does any of you have idea how to fit a parabolic equation to describe that curve? Thanks in advance.

tj.

Statistical Tools

Sun, 08 Jan 2012 12:01:47 +0000

A very interresting site with several statistical tools and you can do it online

(Clinical, Research, Calculators, Probabilities, Distributions, Frequency Data, Proportions, Ordinal Data, Correlation & Regression, t-Tests & Procedures, ANOVA, ANCOVA, Miscellanea)

http://faculty.vassa...assarStats.html

Help with verifying that I'm looking at polymorphisms?

Fri, 06 Jan 2012 15:04:18 +0000

Sorry that I couldn't think of a better sounding title!

Basically I've had to do some Bioinformatic work recently having never ever done or even heard of the subject before, so I'm a complete novice as to how to use all the websites like NCBI, Vega Sanger, BLAST, BLAT... etc.! and I'm finding it really hard to understand exactly what it is that I'm looking at.

I've been trying to find the number of polymorphisms in a particular gene, human TRIM5. I found one website with it on http://www.uniprot.org/uniprot/Q9C035 and simply counted the 'Natural Variants' to come up with a number of 10. However, I would be really grateful if anybody could give me some guidance. Are these natural variants actually the same as polymorphisms? Have I actually managed to include all of the polymorphisms (if they are indeed 'natural variants') or am I accidentally only looking at a subset of polymorphisms?

Just to re-iterate the fact that I really feel out in deep water with this subject and so if you do give any answers please feel free to phrase any reply as if I'm a little bit stupid! I really want to understand what all these things are, feeling lost is a very horrible feeling!

Any help would be MUCH appreciated.

Thanks in advance!

TCGA portal

Thu, 29 Dec 2011 05:39:45 +0000

Can anyone tell me how to read the results of TCGA portal?
what does AgilentG4502A_07 z-score ↓ of 85% mean?

BLAST of ITS4 ITS5 sequence

Mon, 26 Dec 2011 14:54:20 +0000

Hi, I have a question about BLASTing. Sequencing was done for a fungal strain. The PCR primers were ITS4 (reverse) and ITS5 (forward). I used the ITS4 sequence to BLAST and it gave a different results than the ITS5 BLAST; both results are quite close - results showed strain from the same genus; but the top result was different. Did I use BLAST wrongly? Or to rephrase the question, since I have one reverse sequence and one forward sequence, which should I use to BLAST or will need to do anything to the sequence before BLASTing? Thanks in advance!

Comparison of gene structure

Tue, 20 Dec 2011 17:46:54 +0000

Hello everyone,

I have two genomic sequences, and I would like to know if there is any relationship between them regarding the protein coding genes (same proteins?, same order?). They definitely contain mutations on the nucleotide level, so a simple nucleotide BLAST with two sequences as queries is a bit pointless. They are already annotated, so I could theoretically check the corresponding Genbank-Files and look up each and every entry there and do protein BLASTs with the sequences, but that would "a bit" tedious. Moreover, I don't trust automated annotations. My question therefore is: does anybody know a good program that translates two sequences in all reading frames and compares the results?

Thanks for suggestions in advance!

Greetings
Freemason

Guessing candidate miRNAs from mRNA microarray

Sun, 18 Dec 2011 16:56:39 +0000

Hi every one,

There are several articles that have worked on the effect of a certain compound (of interest in my project) on mice. They have results from mRNA microarrays and have identified differentially expressed genes and pathways in the response of mice to the chemical.
Is there a way that I use this information to "guess" the putative miRNA alterations in human response to the aforementioned compound?

Thank you in Advance

repeat masking of gene enriched genomic reads on 454 platform

Fri, 16 Dec 2011 15:58:33 +0000

hi,

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i have sequenced gene enriched genomic reads on 454 platform. After removing reads of organeller genome, i want to assemble the filtered reads on gsAssembler. Being genomic reads, should i repeat mask it prior to assembly? Whether gsAssembler can assemble repeat masked reads?

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KM (India)

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repeat masking of gene enriched genomic reads on 454 platform

Fri, 16 Dec 2011 15:57:53 +0000

hi,

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KM (India)

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Sample Size determination

Tue, 06 Dec 2011 19:35:08 +0000

I am doing dose response analysis (ie probit or logistic) with data that is highly variable. One of my colleuges suggested using Jackknife or Bootstrapping to determine the best sample size. However, I haven't been able to find an example of this in the literature. Does anyone have any suggestions?

BLAST of shRNA sequence

Tue, 06 Dec 2011 18:32:48 +0000

Dear all,

I want to blast my shrna sequence to see if it is specific. I used megablast (NCBI) and nucleotide collection as database. I got many many things other than my gene of interest.

Could you please tell me, how I can determine if my shrna is specific enough to my gene of interest? Is "total score" a good representative for specificity? Should I use nucleotide collection as database or refseq-rna (this one gives less hits)?

Thanks

PAML tree design

Sun, 27 Nov 2011 20:20:20 +0000

I'm new to PAML and phylogenetic analysis in general. I'm running a bunch of alignments through PAML to calculate dN/dS per branch and I need to give it a species tree but I'm not familiar with the parenthetical notation it requires. Can anyone help me out?

The species I'm using are:
Human, Chimp, Orangutan, Gorilla, Mouse, Rhesus Macaque, & Cow

Thanks!