- I recently did a TubeSeq service from Eurofins. (submitted PCR amplicons and forward primer)
- My target was an ITS2 region of some Haemonchus contortus parasite isolates.
- I was doing a BLAST run (for nucleotide) of the sequence FASTA file but BLAST gave no result. MEGA X app also read it as a protein sequence.
- Apparently, it is "a protein sequence" and thus, BLAST cannot find any similar records in the database.
- I am attaching one of the sequences and it does look weird to me. At the moment, I am still awaiting a response from Eurofin's technical team.
- Has anyone come across such an error/faulty result? Or am I missing something here?
- Please help!
Check the quality of the sequence and you will get your answer by the chromatogram.
Abhijeet Singh hi, Abhijeet. Thank you for the answer but can you please elaborate on it?
This looks like an insertion/deletion sequence leading to most bases being 2 different bases at each sequenced base position. Can we see the original .abi sequence file and also a normal .abi sequence file for comparison
Paul Rutland Thank you for your reply.
If it helps, the technical team of Eurofins reached out to me and quote their response:
"had a look at the results and the chromatograms unfortunately show mixed signals and therefore the corresponding FASTA files will show IUPAC bases at these positions wich are then probably misinterpreted by your software which says that the seqeuence belongs to a proein. Nevertheless, the sequences are linked to your DNA samples but the analysis did not yield a nice and clean sequence."
They asked me if my DNA was analysed for quality which I did. Also, the PCR replicates gave clear bands on AGE.
So, is there any way the result can be somehow salvaged or do I have to send it again? I am also attaching a screenshot of one of the abi files,
Any input here would help me a lot, I am fairly new in this area.
Thank you.
I would say that the major peaks could be read ignoring the minor peaks but it would be easier to analyse with an original .abi file of both this dirty sample and a cleanly sequenced sample.
AGE will not distinguish between 2 sequences differing in size by 2% or less so if this was an insertion/deletion of less than a few bases then it would not show on AGE. They are right that a sequence with many iupac mixed bases will be seen as protein not dna but the sequence could still be read by eye then BLASTed
Paul Rutland I appreciate your quick responses. At the moment, I am planning to resend a new batch of samples for sequencing. Hopefully, I will get a better result.
Just one small question again, pardon my ignorance. One of my projects includes an analysis of phylogeny with the isolates our lab has collected. Is there any way these current results can be salvaged and used for it? I understand any tree constructed using this would probably give false result, I would just like to give a try to use the MEGA software.
Any idea on this?
Yes it is a problem reading sequences like this. When sending it for resequencing use the other pcr primer for sequencing then you will have 2 sequences and it will be easier to see the correct base. Many of your mixed bases can be resolved by reading the highest peak base and looking for patterns like if 4 consecutive bases all have a G base at low intensity then it is likely that the Gs are artifacts or maybe the problem is just that the base peak intensities are low and background noise is being pulled up by the analysis software but it is difficult to say without seeing the original sequence file from the company
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