I have sequenced a bacterial 16S rRNA coding region using capillary sequencing and then got the De-Novo assembly (DNA sequence) of the same bacteria (same pure culture). After a blast search I wasn't able to find any hit between these two sequences. Can anyone suggest the reason behind such a disconnect?
which program did you use for assembling? Some programs considered 16S rRNA as a "repeat" and they removed it from the final assembly.
I don't understand, you have sequenced 16S of pure colture bacteria using ion torrent or capillary sequencing (I presume by Sanger procedure)?
16s by sangers, whole genome by ion torrent. MIRA3 and Newbler for assembly
Did you do similarity search of the rRNA sequence against nr database?
yes and found positive result. Very close to the bacteria of similar class
I have no expreience on ion torrent sequences but I have experience on the Illumina data (mixed microbial populations). The most modern tool for ribosomal gene finding on Illumina reads seems to be RNASelector [Lee JH et al. J Microbiol. 2011 49(4):689-91], that use HMMer on reads towards an HMM model of fwd and
rev 16Ss of bacteria and archea. Although the program (in java, with GUI, phuah...) crashed frequently, the rationale is quite clear.
I would do a simple blast search of your RNA sequence obtained by Sanger against all reads from ion torrent. If you get a positive results it would suggest that your assembler filters our ribosomal RNA as suggested by Luca. Then, read a manual for the assembler. You will surprised how much you can learn :)
Ion Torrent is by far the worst sequencing platform in terms of sequencing errors.
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