I ve aligned a list of sequence to a transcript to observe if this sequences are present in the transcript. I produced a BAM file. This is the result of my Alignement.
Left reads:
Input : 27849344
Mapped : 16442 ( 0.1% of input)
of these: 7259 (44.1%) have multiple alignments (56 have >100)
Right reads:
Input : 27849344
Mapped : 15211 ( 0.1% of input)
of these: 8390 (55.2%) have multiple alignments (56 have >100)
0.1% overall read mapping rate.
Aligned pairs: 5975
of these: 3776 (63.2%) have multiple alignments
194 ( 3.2%) are discordant alignments
0.0% concordant pair alignment rate.
~
Now I have to see that I don't count the same read twice, because I have changed the multiple alignment parameters. If a read maps on two different sequences, it is always the same read. I have to count the reads as unique. So how can I made this check?
1 answer
I assume you used tophat2 for this alignment. Refer to this post which explains mapping qualities for tophat2: Tophat2 Mapping Qualities
I suggest that you try to see how many reads show up with following commands (you are just getting counts):
$ samtools view -c -q 30 your.bam
$ samtools view -c -q 40 your.bam
$ samtools view -c -q 45 your.bam
$ samtools view -c -q 50 your.bam
That last command will likely show reads that are uniquely mapped. You can remove -c if you want to capture those to a file.
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Depends on the alignment tool you used and whether or it includes such tags.
Also please use the search function.
I'VE used tophat. I would make this analysis on R
There is no logical overlap between those two. One is a splice-aware alignment program and other is a statistical programming environment.
Sorry, I would to analize the bam file produced with tophat in r environment
What kind of analysis?
A kind of analysis that allows me to count the repetitive elements in the trascripts. Anyway I ve tried a solution. I opened the bam with samtools, and I made unique() to count the unique reads that drop in some elements of interest. For example I made unique to all the reads presents in SINEs
Come ooooooon SeaStar.
You should have known that in order to write and ask for something in biostars you should be awarded with a Pulitzer Prize!!!
Of course we understood what you were talking about...
:-D :-D :-DI am sure about the future reply below ↧ ↧ ↧ ↧ ↧ ↧
Hi,
How does your comment contribute to the discussion? This might be a language gap, but the content of the comment is quite unprofessional. Would you mind editing it so it is relevant to the discussion and framed in a professional manner please?
If you have any criticisms to offer on Biostars, you are welcome to start a Forum discussion post, or better, join us on the biostars slack.
It s just a common observation in many biostars posts flavoured with a pinch of humor.
Of course there are different standards of sense of humor amongst individuals.
You are free to not see the "funny side of things", but as far as I do not use any strong language or an insult, you cannot accuse me for "unprofessionalism".
There are many people here that they are really trying hard to understand some concepts. Criticism is not always the key to help them.
End of story. Please do not add more noise to the post. I won't
Before doing that, I would worry about the
0.1% overall read mapping rate.... Do you think it is normal to have such a low alignment rate ?This is likely because full dataset is being aligned to
a transcript. Not generally recommended but probable explanation for that0.1%figure in this case.I'VE aligned a set of transposon to a transcript to observe if these transposable elements are presents. It is a experimental test. I've used tophat
What exactly are you trying to show? Is the following interpretation correct.
Yes, it is correct. I want to check if the reads don't align 2 or more times the same sequence