filter sequences from rRNA, tRNA

[cascoamarillo]

Hi all,

After the first look, I make a rapid clone survey, to my small RNA library (for Illumina); things seem to be working. But I want to measure the grade of contamination from other sequences, like degraded rRNA, tRNA or even E.coli sequences. I think it could be a good starting point to BLAST my seqs against specific ribosomal RNA databases and/or E.coli database (intead of the whole nt db).
So my question is: where can I find these databases? I'm being looking for a while throught the ncbi/embl but cound't find anything. Or if anyone has a better idea to check out this contamination...

[NicoBxl]

rfam : http://rfam.sanger.ac.uk/

[cascoamarillo]

Thanks for the information!

But I've another question. When I have an Illumina read and I want to perform a filtering from contaminating seqs (eg. E. coli), I mean, to take off those non-desire reads: how can I do that. Is there any program/script for that pourpose?

Thanks!

[NicoBxl]

The best way is to keep the reads that align with your reference genome.

[Gators]

The best way is to keep the reads that align with your reference genome.

That's the best way, but you could always align your reads against e.coli or whatever your putative contamination is, and use the bowtie parameter -un that saves unaligned reads.

[carmeyeii]

That's the best way, but you could always align your reads against e.coli or whatever your putative contamination is, and use the bowtie parameter -un that saves unaligned reads.

Of course. Very clever, thanks!

[carmeyeii]

I am analyzing some Illumina libraries that appear to have a lot of ribosomal RNA contamination.

I'm using Bowtie to align the reads only to a specific set of sequences, and because of the differing amount of rRNA contamination in each sample, each of them maps a different percentage of reads to the dataset (some half of what others map), ranging from 1% to 0.3%.

I wonder if the amount of rRNA contamination in the preparation of a library can have an impact on the apparent expression level of a gene -- even though one normalizes its counts agains the total number of reads that mapped.

What's your opinion in this subject?

Carmen

[alisrpp]

Hi,

I'm trying to use Bowtie, as Gators was suggesting, to clean my raw reads of rRNA contamination but bc it's my first time using Bowtie i'm a little bit lost.
Can anyone suggest me a script for that with the --un parameter?
Thanks,

[alisrpp]

Hi to all,

Finally i could write a bowtie script that is working. To generate my indexes i download all the Porifera rRNA sequences fro NCBI.
Now i have 2 questions:

- As an input, should i use my FASTQ raw reads (without any trimming, either trimming of the adapters or by quality) or it's better if i clip the adapters and filter by quality first and then i try to remove the rRNA contamination?

- I'm trying to print the general statistics using the -t parameter but i guess that because i'm launching the script to a queue i'm not getting anything. How can i obtain the general statistics information?

Time loading forward index: 00:00:00
Time loading mirror index: 00:00:00
Seeded quality full-index search: 00:00:00
# reads processed: 1000
# reads with at least one reported alignment: 699 (69.90%)
# reads that failed to align: 301 (30.10%)
Reported 699 alignments to 1 output stream(s)
Time searching: 00:00:00
Overall time: 00:00:00

Thanks,
Alicia.