I want to find novel and known RNAs and transcripts in a sequence of about 10 KB. What is the most easiest way using bioinformatics tools to start with if that sequence is not well annotated in ensembl and UCSC browsers? Does splices ESTs and RNA sequencing data one option? I am new to bioinformatics, your suggestions are useful for me.
Thanks in advance
I am a bit unclear on what exactly your desired end-product or output would look like. But I might suggest doing multiple sequence alignments and looking for those with high scores. Chances are if this 10KB sequence will have some of those known sequences but they won't match exactly, so I think you want a program that gives you alignment scores and not just simple matches. I use Perl in combination with Clustal to make alignments. Basically, you will need to make .fasta or .aln files with both the 10KB sequence and a known sequence of interest according to those file formats' respective convention. You can use the GUI version of clustal if you are not too programming savvy. If you want to use Perl, here is a script I wrote for aligning a whole directory of .fasta files. It can perform many alignments in one fell swoop. NOTE: you must edit the clustal executable path in the last line (system call) to match its location on your computer for this script to function.
#!/usr/bin/perl
use warnings;
print "Please type the list file name of protein fasta files to align (end the directory path with a / or this will fail!): ";
$directory = <STDIN>;
chomp $directory;
opendir (DIR,$directory) or die $!;
my #file = readdir DIR;
closedir DIR;
my $add="_align.fasta";
foreach $file (#file) {
my $infile = "$directory$file";
(my $fileprefix = $infile) =~ s/\.[^.]+$//;
my $outfile="$fileprefix$add";
system "/Users/Wes/Desktop/eggNOG_files/clustalw-2.1-macosx/clustalw2 -INFILE=$infile -OUTFILE=$outfile -OUTPUT=FASTA";
}
Do you have a linux server or computer or are you relying on web and windows-based programs?
To align RNA-seq reads, people generally use splice read aligners like Tophat, although BLAST would probably work too.
Initially I wrote long response explaining how to do this in Linux but I've just realised that Galaxy might be a much easier solution for a beginner. Galaxy is an online bioinformatics tool with a very user friendly interface; it's particularly designed for beginners. You can sign up and log in at this website: https://main.g2.bx.psu.edu/
There are tutorials on how to do things (see 'Help' menu) but my basic workflow for your experiment would go something like this:
Log into Galaxy
Upload RNA-seq reads, EST reads and 10K genome sequence
In the menu on the left, click to expand "NGS-RNA sequencing", then click "Tophat for Illumina (assuming your RNA-seq reads are Illumina fastq reads)"
Align your RNA-seq reads using Tophat, make sure to select your 10K sequence as the reference genome.
Try aligning your EST reads with one of the programs. I'm not sure how successful this will be, Tophat isn't designed to work with long sequences so you might have to have a bit of a play or be a bit creative to get this working.
Use Cufflinks to create annotation for novel gene models, based on your RNA-seq reads and/or EST sequences.
Regarding viewing the output, I'm not sure what is available for a custom reference sequence on Windows, you might have to do a bit of research. For Linux/Mac, I'd recommend IGV.
Related
Is there a way to edit the raw text from a PDF without any special paid software?
So there are PDFs with highlightable text. I assume that the text is stored somewhere in the file.
I tried to just drag & drop a PDF into vscode but it just showed me unknown characters; even a little of meta text but if I edit the meta-infos, the file gets mostly corrupted.
Apart from that, I could not find any of the text contents of my desired PDF in vscode-editor.
Does someone know if there is a solution like inspecting and changing the source code somehow without a special software? I want to edit the contents; not the meta-infos.
(I use macOS)
The text you see on a pdf page can be constructed in dozens of different ways, actually there are millions of users, using potentially hundreds if not thousands of different methods.
Update
The question is MacOS but for native cross platform you need to work in mime text/pdf to be universally useful. But by way of example how thats possible specifically in windows its possible to write line by line using say cmd here is a snippet of what was a few dozen lines :-)
echo %%PDF-1.0>demo.pdf
echo %%µ¶µ¶>>demo.pdf
echo/>>demo.pdf
for %%Z in (demo.pdf) do set "FZ1=%%~zZ"
echo 1 0 obj>>demo.pdf
echo ^<^</Type/Catalog/Pages 2 0 R^>^>>>demo.pdf
echo endobj>>demo.pdf
echo/>>demo.pdf
For the fuller "Feature Creep"ing of now over more than a 100 lines and counting see
https://github.com/GitHubRulesOK/MyNotes/raw/master/MAKE-PDF.cmd
However although plain text could be the simplest it is rarely used except to prove a conceptual point that it is possible. The rest of the time "Special Software" as you call it (a pdf generator/editor) will be used to compress the file objects, most frequently as different optimal binary streams.
So some text may be scanned pixels whilst other text may be line shapes that look like letters, or at other times plain letters without fonts but a named style, or even letters with the font included (embedded) in the file (the preferred option).
In many ways each page may be built different to the others and thus no two pdfs generally will use the same structure unless like a bank statement using a format that does not change much from month to month, even if the balance wobbles about.
So in summary the tool that will work best is the one that covers every single permutation that Adobe dreamed of, and still keep the result a valid Adobe PDF.
Thus Acrobat PRO 3D is on my shelf (even if not used from one year to the next)
There are many cheaper editors and ones I will use more often for small mods are Tracker Xchange and FreePDF PRO and both have different limitations.
Your choices for MacOS will be more limited thus search for the best you are willing to pay for.
How could I read Garmin's .fit file on Linux. I'd like to use it for some data analysis but the file is a binary file.
I have visited http://garmin.kiesewetter.nl/ but the website does not seem to work.
Thanks
You can use GPSbabel to do this. It's a command-line tool, so you end up with something like:
gpsbabel -i garmin_fit -f {filename}.fit -o csv -F {output filename}.csv
and you'll get a text file with all the lat/long coordinates.
What's trickier is getting out other data, ie: if you want speed, time, or other information from the .fit file. You can easily get those into a .gpx, where they're in xml and human-readable, but I haven't yet found a single line solution for getting that data into a csv.
The company that created ANT made an SDK package available here:
https://www.thisisant.com/resources/fit
When unzipping this, there is a java/FitCSVTool.jar file. Then:
java -jar java/FitCSVTool.jar -b input.fit output.csv
I tested with a couple of files and it seems to work really well. Then of course the format of the csv can be a little bit complex.
For example, latitude and longitude are stored in semicircles, so it should be multiplied by 180/(2^31) to give GPS coordinates.
You need to convert the file to a .csv, the Garmin repair tool at http://garmin.kiesewetter.nl/ will do this for you. I've just loaded the site fine, try again it may have been temporarily down.
To add a little more detail:
"FIT or Flexible and Interoperable Data Transfer is a file format used for GPS tracks and routes. It is used by newer Garmin fitness GPS devices, including the Edge and Forerunner." From the OpenStreetMap Wiki http://wiki.openstreetmap.org/wiki/FIT
There are many tools to convert these files to other formats for different uses, which one you choose depends on the use. GPSBabel is another converer tool that may help. gpsbabel.org (I can't post two links yet :)
This page parses the file and lets you download it as tables. https://www.fitfileviewer.com/ The fun bit is converting the timestamps from numbers to readable timestamps Garmin .fit file timestamp
I like reading the PoC||GTFO issues and one thing I found remarkable when I first discovered it, was the "polyglot" nature of their PDF files.
Let met explain: when you consider for example their 8th issue, you may unzip files from it; execute the encryption they are talking about by running it as a script and even better(worse?) with their 9th issue you can even play it as a music file!
I'm currently in the process of writing small scripts every week and writing each time a little one page PDF in LaTeX to explain the said scripts. So I would really enjoy being able to create the same kind of PDF files. Sadly they explained (partly) in their first issue how to include zip files, but they did so through three small sketches of cmd lines without actual explanations.
So my question is basically :
how can one create such a polyglot PDF file containing stuff like a zip as well as being a shell script which may be run using arguments just like normal scripts?
I'm asking here about the process of creation, not just an explanation of how this is possible. The ideal way for me would that there are already some scripts or programs allowing to create easily such PDF files.
I've tried to search the net for the keywords "polyglot files" and others of the kind and wasn't able to find any useful matches. Maybe this process has another name?
I've already read the presentation by Julia Wolf which explains how things works, but I sadly haven't had time to apply the knowledge there to real world, because I'm sadly not used to play with file headers and the way a PDF is constructed.
EDIT:
Okay, I've read more and found the 7th edition of PoC||GTFO to be really informative concerning this subject. I may end up being able to create my own scripts to do such polyglot PDF files if I have some more time to consider it.
I played around with polyglots myself after attending Ange's talks and also talking to him in person. You really need to understand the file formats to be able to nest them into each other.
However, long story short, here are some links I found extremely useful for creating polyglots:
Some older Google Code Trunk
PoC of the polyglot stuff
Especially the second link (to github) will help you creating polyglots, but also understanding how they are working and how they are implemented. Since it is mostly Python stuff and very well / clean written, it is very useful and easy to follow.
I feel dissecting some file formats would be a good place to start. You can find many file format specifications for different file types through Google, but they can be a tough read and will likely take you some time to translate into whatever language you are using.
PDF: https://www.adobe.com/content/dam/acom/en/devnet/pdf/pdfs/PDF32000_2008.pdf
ELF: https://www.cs.cmu.edu/afs/cs/academic/class/15213-s00/doc/elf.pdf
ZIP: http://kat.sdf.org/zip_file_format.txt
The language(s) you select will need a way to read and write raw bytes (not just ascii alphanumeric), so perhaps C would be good for more direct access to memory. Some Python tricks could help with open sourcing the scripts easily.
To dissect the files, you may want to build a tool kinda like https://github.com/kvesel/zipbrk/ to take them apart, then put them all back together in a polyglot format. For example, zip does not require the section headers to be at the start (or even contiguous for that matter), and PDF magic number can appear in multiple places within the file as well. I also believe I recall a polyglot tool being included in one of the PoC||GTFO publishings (maybe issue 8 or 2??) as a polyglot in the pdf file.
Don't forget the hackers bible! :)
https://nostarch.com/gtfo
I am a novice Go lang programmer,trying to learn Go lang features.I wanted to split a large csv file into multiple files in GO lang, each file containing the header.How do i do this? I have searched everywhere but couldnt get the right solution.Any help in this regard will be greatly appreciated.
Also please suggest me a good book for reference.
Thanking You
Depending on your shell fu this problem might be better suited for common shell utilities but you specifically mentioned go.
Let's think through the problem.
How big is this csv file? Are we talking 100 lines or is it 5G ?
If it's smallish I typically use this:
http://golang.org/pkg/io/ioutil/#ReadFile
However, this package also exists:
http://golang.org/pkg/encoding/csv/
Regardless - let's return to the abstraction of the problem. You have a header (which is the first line) and then the rest of the document.
So what we probably want to do (if ignoring csv for the moment) is to read in our file.
Then we want to split the file body by all the newlines in it.
You can use this to do so:
http://golang.org/pkg/strings/#Split
You didn't mention but do you know how many files you want to split by or would you rather split by the line count or byte count? What's the actual limitation here?
Generally it's not going to be file count but if we pretend it is we simply want to divide our line count by our expected file count to give lines/file.
Now we can take slices of the appropriate size and write the file back out via:
http://golang.org/pkg/io/ioutil/#WriteFile
A trick I use sometime to help think me threw these things is to write down our mission statement.
"I want to split a large csv file into multiple files in go"
Then I start breaking that up into pieces but take the divide/conquer approach - don't try to solve the entire problem in one go - just break it up to where you can think about it.
Also - make gratiutious use of pseudo-code until you can comfortably write the real code itself. Sometimes it helps to just write a short comment inline with how you think the code should flow and then get it down to the smallest portion that you can code and work from there.
By the way - many of the golang.org packages have example links where you can literally run in your browser the example code and cut/paste that to your own local environment.
Also, I know I'll catch some haters with this - but as for books - imo - you are going to learn a lot faster just by trying to get things working rather than reading. Action trumps passivity always. Don't be afraid to fail.
Here is a package that might help. You can set a necessary chunk size in bytes and a file will be split on an appropriate amount of chunks.
I have a bam file and use bioperl (Bio::DB::Sam) to work with it.
Now i wanted to ask if there is any possibility to add tags to alignments in this File?
i use
my $iterator = $bam->features(-iterator => 1,
-flags => {M_UNMAPPED=>0});
while (my $align = $iterator->next_seq) {
...
}
to loop through the aligned reads. Now i am searching vor anthing like
$align->addTag(key=>value)
bye
BedTools has a means of annotating BAM files called TagBam
http://biostar.stackexchange.com/questions/9552/basic-bam-file-annotation
https://github.com/arq5x/bedtools
Short answer, no.
It might be possible to do it to the raw SAM and then convert it back to BAM. I've recently been writing a C++ api for BAM and came across this issue myself. The problem is the underlying c structures representing this information are all tightly byte packed and take up a specific amount of memory. Sometimes they might have a little more than they need, but other times they have just the right amount. Adding to these structures would, in most cases, exceed the available memory to them.
So, if I were you I would write out a new SAM file with your additional tags in it and convert it to BAM using samtools