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Old 05-11-2016, 07:12 AM   #1
bio_informatics
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Default BLAST: no output

Hi,

BLAST 2.2.30+

I've 16 nucleotide base sequences, which I'd like to use to gather pairwise identity against another FASTA file.

Code:
makeblastdb -in barcode.fasta -dbtype nucl -out barcode.fasta -title newdb
This works fine, I get 3 files ending in with .nhr, and likewise.

For some reason(s) I don't get desired results using this DB.
I go ahead and BLAST my input file against itself.

Code:
blastn -query barcode.fasta -out ./3blasted_seq.txt -subject barcode.fasta
I get no hits everywhere.

with -outfmt flag, and option 6 I get empty file.

Tried with ' and " with -outfmt 6 to get desired results, but in vain.

I used -dust flag with option no I see no change in output

I'm running out of options to try to have desired results.
Any help will be greatly appreciated.

I've looked for help at:

http://www.ncbi.nlm.nih.gov/books/NBK279675/

http://seqanswers.com/forums/showthread.php?t=59798

https://www.biostars.org/p/87689/

https://www.biostars.org/p/42687/

https://www.biostars.org/p/121033/
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Old 05-11-2016, 07:35 AM   #2
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Found solution, finally.

https://www.biostars.org/p/47203/

-task blastn-short

is crucial
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Old 05-11-2016, 07:41 AM   #3
GenoMax
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Have you tried -task blastn-short? 16 bp may be a bit short in any case.

What exactly are you trying to do (some clues are there in the file names)?
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Old 05-11-2016, 07:46 AM   #4
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Quote:
Originally Posted by GenoMax View Post
Have you tried -task blastn-short? 16 bp may be a bit short in any case.

What exactly are you trying to do (some clues are there in the file names)?
Hi GenoMax,
Thank you for your reply.
Yes, I used this flag, posted relevant URL where I found solution, a reply ahead of you.

I'm working with 16S data, which uses complicated process, and sequences barcodes, and amplicon separately. At QIIME demultiplex step I lose ~33% of my data due to barcode not found.
I've 2 bar-codes: one from lab technician, saying these belong to our samples.
And another set, which I find that got sequenced.

We're trying to drill down if there has been any barcode that has been sequenced or not as per our input mapping bar-codes.
Sorry if that sounds confusing.

Thanks.
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Old 05-11-2016, 07:54 AM   #5
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It is not confusing. We do run into this periodically. Mistakes happen/wrong libraries get pooled (you get the idea).

Thing to keep in mind is what is in the sequence data is real (if that is the correct word). If that data is not matching what the technicians claim should be there then you need to be on guard. Remaking the library may be the only option (unless you have some other way to ID the samples).
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Old 05-11-2016, 08:03 AM   #6
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Quote:
Originally Posted by GenoMax View Post
It is not confusing. We do run into this periodically. Mistakes happen/wrong libraries get pooled (you get the idea).

Thing to keep in mind is what is in the sequence data is real (if that is the correct word). If that data is not matching what the technicians claim should be there then you need to be on guard. Remaking the library may be the only option (unless you have some other way to ID the samples).
Hi GM,
Yes, I get what you're saying.
Wow, we both got what we both were saying seldom has that happened with me.

Sequencing center sits in a different location, any direct criticism on library (or anything lab related) needs to be supported with numbers on our end.
Thus, trying to come up with these % numbers, and relevant counts so we can put it politely.

Thank you again for your time, and reply.

Cheers!
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Old 05-11-2016, 08:16 AM   #7
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So you are not looking at gross disparity (you expect one index but it is something else in sequence) but 1 bp differences?

Difference in %'s (read #) in a pool are going to be dependent how well the libraries were quantified and pooled.
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Old 05-11-2016, 08:28 AM   #8
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Quote:
Originally Posted by GenoMax View Post
So you are not looking at gross disparity (you expect one index but it is something else in sequence) but 1 bp differences?

Difference in %'s (read #) in a pool are going to be dependent how well the libraries were quantified and pooled.
Hi GM,
I agree with what you said. I really doubt data on my end, but I'm limited to my thoughts, and opinions.
This data lose of 33% is something sequencing center is unaware of, and weighing on the data at hand which get generated. The quality is really poor (QC gives no quality of reads after 290-ish).
V3-V4 chemistry has of late (2 years) been under constant vigilance by Illumina, and researchers.

Data I've is Mi-Seq. 2*17G file Illumina paired end. 300 bp length
I took initial 100K reads to get something going. I'm aware that those 100K doesn't represent entire data.
I know this sounds weird.

Thank you again for your reply.
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Last edited by bio_informatics; 05-11-2016 at 08:40 AM.
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