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Old 05-07-2014, 02:20 AM   #1
jyuems
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Location: Finland

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Smile Bowtie2: rRNA data from Silva fail to align with Illumina PE reads

Hi!

Could you help me with my long list of problems? I have Illumina HiSeq paired-end RNA data from an unmodel organism. The reads have a lot of rRNA and other sources of contamination and they have quite low quality scores.

I'm trying to filter out the rRNA out of the samples using the whole length shotgun sequences from a closest model species. I can't use the species whose rRNA genes we have found from the data because they have only partial sequences available. So I downloaded the rRNA SSU and LSU r117 sequences from Silva as FASTA without gaps and decompressed the .tar.gz files into FASTA before usage. Should I use some other format than FASTA without gaps?

I created the index out of the Silva-files and aligned the reads using Bowtie2 using simplest possible commands:

Quote:
bowtie2-build C_brenneriSSU.fasta C_brenneriSSU_index
Quote:
bowtie2-align -x C_brenneriSSU_index -1 ove2_R1.fastq -2 ove2_R2.fastq -S result.fastq
But nothing is aligning. Why is that? The reads are untrimmed because I've read that it's best for Bowtie2 to use the original reads.

We have also tried to align some example rRNA-sequencefiles and the Silva-files with consensuses of contigs constructed from combined samples gained from CLC assembly but they won't align with the rRNA datas with Bowtie2 either. Any ideas why not?

And most importantly, what should I do to find the rRNA sequences? I'm familiar with extracting the unmapped reads by using samtools so it's basically just the alignment that seems to be the problem.

Last edited by jyuems; 05-07-2014 at 04:12 AM.
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Old 05-07-2014, 02:30 AM   #2
jyuems
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This is the result from Bowtie2:

Quote:
36906390 pairs aligned 0 times concordantly or discordantly; of these:
73812780 mates make up the pairs; of these:
73812780 (100.00%) aligned 0 times
0 (0.00%) aligned exactly 1 time
0 (0.00%) aligned >1 times
What could be going wrong here?
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Old 05-07-2014, 02:57 AM   #3
guptavipin142
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Hi Jyumes...

I am also new but all i can suggest that use MGRAST for rRNA Annotation. There is a paper in plos for this study.
Also can download rRNA database for prokaryotes and eukaryote and perform Blast (all vs all) for tabular out put.
this will solve the rRNA reads.

Also i would like to ask have you used any rRNA cancellation method during RNA isolation otherwise there is uptp70% rRNA reads contamination in your sequences.
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Old 05-07-2014, 04:26 AM   #4
jyuems
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Thank you guptavipin142! I will give the MGRAST a go.

Yes, the samples were attempted to be purified from rRNA twice during isolation but according to BLAST results there are still a many rRNA sequences. Also according to FastQC report there are a lot of overrepresented sequences present in the samples.
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