Entering edit mode
5.7 years ago
young_bioinformatician
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240
Hi everyone,
My problem is 16s RNA results. I have some 16s sequences, forward and reverse as FASTA. I directly made a blast in ncbi. However, the results shows that many different species have same query cover, e-value, and perc. identity. Which species should I choose from these results ?
Also, I have maldi-tof results which means presents identify of species in a way of chemical process. The results of Maldi-tof are in contradiction with the results of 16s RNA.
So, is it possible that 16s RNA sequences could not be clean because of this contradictory results ?
Regards,
Adding some more questions: Is your goal only classification of the sequences without OTU clustering to compare to the maldi tof results? Why exactly did you use sanger sequencing? Where do you find discrepancies between the results?
Given your pipeline, I am not surprised by the differences you see, you probably can't use the blast result without further processing (like taxonomic binning - 16S rRNA sequencing is typically limited to genus level) Depending on the conservation level between genera, you might not even reach genus level accuracy, so you're blast might show what you're looking for but on a far lower line...
@Carambakaracho: My goal is to reveal identification of species from sanger sequencing. And yes, it is without OTU clustering. I do not have any pipeline for this purpose. I did make blast from ncbi. After this, Some 23S rRNA sequences of species were given me. I also checked them. But, as i said above comment, Some articles and reviews mentioned that Sanger sequencing is not reliable for identification. Therefore, We will use NGS for this issue.
Normally, as I know, we can make blast using 16S sequencing or 23S sequencing, which are NGS products, for identification. Am I right ?
sure, you can. You will always find a result with blast, especially when using conserved sequences. All I can do is raise your awareness to the fact that it's not exactly standard (as genomax wrote, too). This means the blast approach comes advantages like you have it installed and know it, but also limitations, like more complicated interpretation, less community acceptance, etc...
You should use Qiime or Mothur for 16s RNA analysis. They are standard methods of doing this type of analysis.
I will also check it you said. But I dont think so this type of analysis. Because Qiime or Mothur make a cluster analysis, am I right ?
clean the fastq fille by fastqc, create a phylogenetic tree, in certain case the 16s sequences is not suitable to identify microorganismes, when you made a blast, are you selected the type of microorganisme to minimize the result what is your microorganisme ? malditof result interpreatation is everything depends on updating the database
Actually, I have sanger sequencing fasta files. I do not have fastq files. Normally, they were ab1 files. But I converted them to fasta files. Basically, they were PCR products, not NGS products.
This is critical information and should have been added to the original question. What kind of an experiment is this? What is the significance of the maldi-TOF data?
Sorry for late response. I was very busy after posting. Actually, I do not have any experience about maldi-TOF data because of relating with chemical experiment. Because my role of the experiment is to make bioinformatics analysis. However, maldi-TOF is a result which determines the identification of species as i know.
About sanger sequencing, I read some articles and some reviews. Many scientists says that we should not trust it because of mixture of species during PCR.
We compared both of results, and thus we produced one results from these two results for now. Then, We will use NGS for this issue.
@nora: One does not "clean the fastq fille by fastqc". FastQC only does quality analysis of fastq data. We can't create a phylogenetic tree from fastq data. Moving this to a comment since it is not an answer for original question.
http://edwards.sdsu.edu/cgi-bin/prinseq/prinseq.cgi
uploaad your fasta file here to evaluate the quality of sequencing
Don't add new answers unless they provide a new answer to the original question. One can't use a fasta file to evaluate quality of sequencing since fasta file only contains sequence.