<p>I'm in the following situation and I came here to see if I could get some help.</p>
<p>I'd like to use IgBLAST for an analysis on some Alpaca antibody data, so I received some FASTA sequences from a colleague, <code>imgt_gapped_alpaca_IGHV.fasta</code>, <code>imgt_gapped_alpaca_IGHD.fasta</code>, etc.</p>
<p>I used the <code>edit_imgt_file.pl</code> tool to appropriately structure each <code>.fasta</code> file, and to make a BLAST database for each (V, D, J) segment:</p>
<pre><code># Process V segment sequences with edit_imgt_file
edit_imgt_file.pl \
~/VHH/data/imgt-data/imgt_gapped_alpaca_IGHV.fasta &gt; ~/ncbi-igblast-1.21.0/internal_data/alpaca/imgt_alpaca_ig_v.fasta

makeblastdb -parse_seqids -dbtype nucl \
    -in ~/ncbi-igblast-1.21.0/internal_data/alpaca/imgt_alpaca_ig_v.fasta \
    -out ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_V
</code></pre>
<p>After doing that for each of the segments, I went to put all the pieces together and perform the following query:</p>
<pre><code>igblastn \
    -germline_db_V ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_V \
    -germline_db_D ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_D \
    -germline_db_J ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_J \
    -auxiliary_data ~/ncbi-igblast-1.21.0/optional_file/alpaca_gl.aux \
    -domain_system imgt -ig_seqtype Ig -organism alpaca \
    -outfmt '7 std qseq sseq btop' \
    -query ~/VHH/data/ALPACA1-BC1.fastq \
    -out ~/VHH/data/igblast-out/ALPACA1-BC1.fmt7
</code></pre>
<p>Unfortunately, the following error is returned to me:</p>
<pre><code>BLAST query/options error: Germline annotation database alpaca/alpaca_V could not be found in [internal_data] directory
Please refer to the BLAST+ user manual.
</code></pre>
<p>I've searched the internet high and low, but this appears to be a bit of a niche problem, and the BLAST+ user manual promised a little more than it was able to deliver in terms of helpful suggestions.</p>
<p>Any questions/comments/ideas/diagnostics would be very welcome-- if you see any low hanging fruit, please let me know-- thank you so much for your time!</p>

CC BY-SA 4.0
2023-08-17T15:31:36.320Z
What you have looks like the right idea to me.  (I use igblastn regularly for human and rhesus macaque antibodies with custom DB+aux files, but not with a custom organism label itself.)  What files do you see in your alpaca db dir for V/D/J?  Are both igblastn and makeblastdb definitely coming from igblast 1.21.0?  (Any chance something's mismatched between those two binaries, I wonder?)  And where did your alpaca auxiliary file come from?  You could maybe drop the aux part to simplify things while troubleshooting.
Jesse
CC BY-SA 4.0
2023-08-17T15:38:43.787Z
Actually I get the same error if I just change my human or rhesus commands to some arbitrary organism name.  Did you also create the FWR/CDR file like it talks about [here](https://ncbi.github.io/igblast/cook/How-to-set-up.html#procedure-to-set-up-igblast-for-custom-organism)?  I guess all of my questions really boil down to, what's in your `~/ncbi-igblast-1.21.0/internal_data/alpaca` directory?
Jesse
<blast><blastn><makeblastdb>
IgBLAST Error: Germline annotation database could not be found in [internal_data] directory
# IgBLAST Error: Germline annotation database could not be found in [internal_data] directory

(Asked by: username_0 on 2023-08-16)

I'm in the following situation and I came here to see if I could get some help.
I'd like to use IgBLAST for an analysis on some Alpaca antibody data, so I received some FASTA sequences from a colleague, imgt_gapped_alpaca_IGHV.fasta, imgt_gapped_alpaca_IGHD.fasta, etc.
I used the edit_imgt_file.pl tool to appropriately structure each .fasta file, and to make a BLAST database for each (V, D, J) segment:
# Process V segment sequences with edit_imgt_file
edit_imgt_file.pl \
~/VHH/data/imgt-data/imgt_gapped_alpaca_IGHV.fasta > ~/ncbi-igblast-1.21.0/internal_data/alpaca/imgt_alpaca_ig_v.fasta

makeblastdb -parse_seqids -dbtype nucl \
    -in ~/ncbi-igblast-1.21.0/internal_data/alpaca/imgt_alpaca_ig_v.fasta \
    -out ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_V

After doing that for each of the segments, I went to put all the pieces together and perform the following query:
igblastn \
    -germline_db_V ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_V \
    -germline_db_D ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_D \
    -germline_db_J ~/ncbi-igblast-1.21.0/internal_data/alpaca/alpaca_J \
    -auxiliary_data ~/ncbi-igblast-1.21.0/optional_file/alpaca_gl.aux \
    -domain_system imgt -ig_seqtype Ig -organism alpaca \
    -outfmt '7 std qseq sseq btop' \
    -query ~/VHH/data/ALPACA1-BC1.fastq \
    -out ~/VHH/data/igblast-out/ALPACA1-BC1.fmt7

Unfortunately, the following error is returned to me:
BLAST query/options error: Germline annotation database alpaca/alpaca_V could not be found in [internal_data] directory
Please refer to the BLAST+ user manual.

I've searched the internet high and low, but this appears to be a bit of a niche problem, and the BLAST+ user manual promised a little more than it was able to deliver in terms of helpful suggestions.
Any questions/comments/ideas/diagnostics would be very welcome-- if you see any low hanging fruit, please let me know-- thank you so much for your time!

**Tags:** blast, blastn, makeblastdb
> **Comments:**
> **username_1**: What you have looks like the right idea to me.  (I use igblastn regularly for human and rhesus macaque antibodies with custom DB+aux files, but not with a custom organism label itself.)  What files do you see in your alpaca db dir for V/D/J?  Are both igblastn and makeblastdb definitely coming from igblast 1.21.0?  (Any chance something's mismatched between those two binaries, I wonder?)  And where did your alpaca auxiliary file come from?  You could maybe drop the aux part to simplify things while troubleshooting.  
> **username_1**: Actually I get the same error if I just change my human or rhesus commands to some arbitrary organism name.  Did you also create the FWR/CDR file like it talks about [here](https://ncbi.github.io/igblast/cook/How-to-set-up.html#procedure-to-set-up-igblast-for-custom-organism)?  I guess all of my questions really boil down to, what's in your `~/ncbi-igblast-1.21.0/internal_data/alpaca` directory?  

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## 0 answers

