Easier than watching TV
I am still searching a good software to read my sequences....yes I need to modify them when I notice some gaps into the automatic reading!
A very nice software is represented by FinchTV (for download: http://www.geospiza.com/Products/finchtv.shtml )
As usual, I am interested in softwares able to work on different operative systems and Finch is usefull for Mac, Win and Linux users.
With a very easy drag and drop system it is possible to open ABI files and resize the chromatogram peacks. Then, I could directly modify my sequences, selecting bases and overwriting them. Moreover, with the menu Edit -> Insert before base, I insert the the skiped bases.
Finally, it is easy to analyse the reverse primers too: clicking on the reverse complement button you already have the correct reading of your sequence.
And finally, it is very convenient the BLAST's link at Edit -> BLAST sequence
The deeping reading of DNA sequences
"At the deepest level, all living things that have ever been looked at have the same DNA code."
This is the Richard Dawkins sentence that is ringing in my mind while I am searching to read my sequencing chromatograms. In fact, unfortunately I have to search the sequence of some splice variants, namely something that makes the original sequence shorter than the original one. So, I am looking at sequences with not the same DNA code!
My experimental path was long: first I have fought with the PCR protocols. In fact, I have worked (as usual!) with an impossible gene and unlikely reagents and conditions. At the end I obtained clean bands but now I have to justify the presence of additional and unespected bands!!
So, after the sequencing of my PCR products, I need to analyse the sequencing chromatograms but I really don't want to do it manually! It's so boring and I need the help of someone else!
I try to find a software, but the only one present in my reserach institute is a proprietary product and it is jealously garded!
Nicely, I found that GENtle could help me again!
In fact, it is possible to directly import the target sequence from the web interface. Then, I am able to import my sequencing results in AB1 format with the menu File -> Import.
The chromatograms are directly opened in the "sequence" section.
Analysing the chromatograms and following View -> Edit sequences, I modify them substituting some unrecognized bases. In fact, computer programs make mistakes and I need to manually double-check the interpretation of the primary data. Unlikely, the editing work is only in overwrite mode...
Moreover, the program does not mention the parameter "signal" that is important for the evaluation of sample noise (AKA Check the 'Signal Intensity' numbers for your chromatogram, in order to know if your peak are uninterpretable).
If you feel alone in this boring work, I find also "someone" that reads me the sequence, choosing View->read sequences aloud.
Finally, in Tools -> Sequencing Assistent I open a window to choose the sequence to align and I obtain the right location of my fragment on the total cDNA strand!
The chromatograms are directly opened in the "sequence" section.
Analysing the chromatograms and following View -> Edit sequences, I modify them substituting some unrecognized bases. In fact, computer programs make mistakes and I need to manually double-check the interpretation of the primary data. Unlikely, the editing work is only in overwrite mode...
Moreover, the program does not mention the parameter "signal" that is important for the evaluation of sample noise (AKA Check the 'Signal Intensity' numbers for your chromatogram, in order to know if your peak are uninterpretable).
If you feel alone in this boring work, I find also "someone" that reads me the sequence, choosing View->read sequences aloud.
Finally, in Tools -> Sequencing Assistent I open a window to choose the sequence to align and I obtain the right location of my fragment on the total cDNA strand!
The boring count of nuclei and other particles
Have you ever stained nuclei with DAPI or HOECHST in order to get the focus of your confocal image? I am sure that you do it everytime you choose to see something with fluorescence or cofocal microscope.
There are a lot of examples in which we need to count the amount of nuclei that are present in the field of the image. For istance, I have assisted to an oral communication of a friend of mine in which there was a lot of confocal images with nuclear DAPI staining and the aim of the work was exactly the nuclear counting. After the communication I asked my friend about the counting method and he told me, with a boring face, that he manually counted them several times....
So I decided to write about a very useful software able to automatically count particles and so -why not- also our nuclei!
ImageJ is a very useful software that can really help us. You can freely download it at http://rsbweb.nih.gov/ij/download.html.
Choosing File -> Open it is possible to open our file containing the image directly obtaining from the microscope.
In order to convert the RGB image in greyscale with: "Edit" -> Options -> conversion to scale when converting and then with Image -> Type -> 16-bit.
Our image is ready to the analysis. However we need to fix a threshold in order to exclude the noising background of the image: Image -> Adjust -> Threshold followed by Process ->Subtract background e Apply.
If we are able to distinguish some artifacts (like two nuclei that seems to be joint together, we can manually separate them with Process -> Binary -> Watershed.
And now, we can tell our software to count the particles that it can distinguish: click on analyse -> Analyze particles to open another window controlling the parameters of our analysis.
The window requests are: 1) "Which is the minimum dimension of your particles (in pixel)?" If we chose the settled 0-Infinity, we just wants to count all the highlighted nulei.
2) "What kind of particles shape have I to chose during my count?" Modifying Circularity we can correctly answer: choosing 1 we will only count the circular shaped particles. Instead setting 0, we will consider only the stretched particles.
Finally, we can check our results clicking on Show outline: in this way we obtain a new image of our particles with all the counted particles: a very useful tool to see what was missed by the software!
Display results shows us the counting results in another window, while Summarize resumes all the informations about our image. Add to manager is a way to save the counting results: in this way you can use the image for further analysis.
There are other interesting functions: “Exclude on Edges” and “Include Holes”that allow us to choose what kind of particles we want to include in our counting process.
Few days ago I had to prepare (really at last minute) an oral communication for a congress. I was searching an image able to easly represent my counting results.So, after the counting analysis, I chose Analyse -> Plot Profile and the result was only perfect: it very well represented intesity and density of mu spots.
Look at it with your eyes!
There are a lot of examples in which we need to count the amount of nuclei that are present in the field of the image. For istance, I have assisted to an oral communication of a friend of mine in which there was a lot of confocal images with nuclear DAPI staining and the aim of the work was exactly the nuclear counting. After the communication I asked my friend about the counting method and he told me, with a boring face, that he manually counted them several times....
So I decided to write about a very useful software able to automatically count particles and so -why not- also our nuclei!
ImageJ is a very useful software that can really help us. You can freely download it at http://rsbweb.nih.gov/ij/download.html.
Choosing File -> Open it is possible to open our file containing the image directly obtaining from the microscope.
In order to convert the RGB image in greyscale with: "Edit" -> Options -> conversion to scale when converting and then with Image -> Type -> 16-bit.
Our image is ready to the analysis. However we need to fix a threshold in order to exclude the noising background of the image: Image -> Adjust -> Threshold followed by Process ->Subtract background e Apply.
If we are able to distinguish some artifacts (like two nuclei that seems to be joint together, we can manually separate them with Process -> Binary -> Watershed.
And now, we can tell our software to count the particles that it can distinguish: click on analyse -> Analyze particles to open another window controlling the parameters of our analysis.
The window requests are: 1) "Which is the minimum dimension of your particles (in pixel)?" If we chose the settled 0-Infinity, we just wants to count all the highlighted nulei.
2) "What kind of particles shape have I to chose during my count?" Modifying Circularity we can correctly answer: choosing 1 we will only count the circular shaped particles. Instead setting 0, we will consider only the stretched particles.
Finally, we can check our results clicking on Show outline: in this way we obtain a new image of our particles with all the counted particles: a very useful tool to see what was missed by the software!
Display results shows us the counting results in another window, while Summarize resumes all the informations about our image. Add to manager is a way to save the counting results: in this way you can use the image for further analysis.
There are other interesting functions: “Exclude on Edges” and “Include Holes”that allow us to choose what kind of particles we want to include in our counting process.
Few days ago I had to prepare (really at last minute) an oral communication for a congress. I was searching an image able to easly represent my counting results.So, after the counting analysis, I chose Analyse -> Plot Profile and the result was only perfect: it very well represented intesity and density of mu spots.
Look at it with your eyes!
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