I was using Newbler/Ray to assemble MiSeq 250bp reads from single cell library. According to the fact that I don't know the sequencing direction, I am trying both ways - complementary sequence to 16S rRNA gene and reverse complementary. RNAs were not extracted, it was DNA sequencing from single sorted cell. While searching, I was using E-value of 1.
Dear Anna, we would need more information on this.
E.g: what is the search direction? Are you searching 16S rRNA sequences against your assembly or the other way round?
Are you taking splicing into account?
How much error do you tolerate in the finding/aligning?
Or is it that you just cannot find the 16S rRNA in your cell?
Were RNAs extracted?
Please consider these and other issues that may help us answer the question.
If the organism has multiple rRNAs, the rRNA collapse into a single contigs with higher coverage than the genome, so look for it in the degen contigs, or your assemblers equivalent of degen contigs.
Dear Anna,
We normally use a query sequence from a similar bacterium and blast all assemblies/contigs. After location of areas containing 16S rRNA genes, which are then verified by mapping of reads onto them. Quite often there are several 16S genes, and so more than one contig. Despite common belief that these genes are identical within a genome there are often one or few base differences. This raises a question on validity of phylogenetic trees based on a particular 16S rRNA copy ... Apparently, the trees may be different depending on which one is taken for analysis.
Regards,
Andrey
Julie, what do you mean under "degen contig"? Is it possible that rRNA gene was collapsed between different contigs and misassemled?
Andrey, I think I am confused with expression "mapping the reads onto them". What do you mean, taking raw unassembled reads from sequencing and mapping them to the assembled database of contigs? Are you using MAQ or other alignement programs?
Hi Anna. I'm using RNAMMER to find 16S in my cyanobacterial contigs. Here's the link:
http://genome.cbs.dtu.dk/services/RNAmmer/
Hope it helps!
Dear Julie, you just made my day! I have been wondering for weeks why I only found 1 16S copy in my assembly, when I should have found 5. Can't believe we didn't think of this. The coverage of the contigs where I found my one copy is indeed much higher than that of other contigs.Thank you!
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