Problem using a custom blast database in tblastx

Question

[arronar]

Hello.

I tried to create a custom DB for blast.

I downloaded fasta files from NCBI from an SRA project and then run this command

makeblastdb -dbtype nucl -in sra_data-DB.fasta -input_type fasta -out plant_Transcriptome_DB -title plant_Transcriptome_DB

and all seemed that went okey.

Building a new DB, current time: 06/18/2014 17:22:49
New DB name: plant_Transcriptome_DB
New DB title: plant_transcriptome_DB
Sequence type: Nucleotide
Keep Linkouts: T
Keep MBits: T
Maximum file size: 1000000000B
Adding sequences from FASTA; added 411965 sequences in 55.2244 seconds.

Three files created. (nhr,nin,nsq)

After this, i run a test with blastdbcmd to see if everything went right.

blastdbcmd -db plant_Transcriptome_DB.nsq -info

and returned me this:

Database: plant_transcriptome_DB
411,965 sequences; 107,369,081 total bases

Date: Jun 18, 2014  5:22 PM   Longest sequence: 999 bases

Volumes:  /*/*/*/*/*/*/*/plant_Transcriptome_DB

Ok! lets run tblastx and see what is happening:

tblastx -query contig2-query.fasta -db plant_Transcriptome_DB -out output -html

Returns :

Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 2: 6, 8, 13, 33, 43-44, 47-48, 50, 55-56, 58-59, 68-70, 72, 81
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 3: 7, 16, 18, 20-22, 26-28, 30, 35, 39, 41-43, 45, 47, 49, 54, 56-57, 59-60, 63-64, 66-67, 69, 71, 79-80, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 4: 7-9, 14, 16, 18, 22, 25-27, 34, 36, 41-43, 45-46, 53-55, 57, 61, 65, 67, 70, 77, 80-81, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 5: 1, 7, 9, 12, 14, 16, 21, 23, 25, 30, 36, 39, 41-42, 46, 51, 53, 57, 60, 67, 69-70, 73-74, 78-79, 81-82
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 6: 1-3, 17, 21, 29, 31, 34, 38-39, 42-43, 47, 53-55, 59, 61, 63, 65-66, 72, 74-77, 79-80
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 7: 1-2, 5-8, 12-13, 16, 18, 20, 23-28, 31, 34-35, 42-45, 52, 54, 60, 62, 67, 70-71, 75-76, 78
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 8: 1, 4-5, 8-9, 16, 19, 30-32, 36, 39-42, 48, 51-52, 56-57, 62-63, 67-69, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 9: 1, 5-7, 12, 14, 17, 19-22, 24-25, 27-28, 30-31, 33, 36-40, 46-47, 50-51, 54, 57-59, 63, 65, 71, 73, 75, 80-81, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 10: 2-3, 5, 7, 9, 15, 17, 25-26, 31, 34, 36-39, 42, 45-47, 50, 57, 62, 64, 69, 72, 76-78
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 11: 3, 7, 10, 12, 15, 17, 23-25, 31, 33, 36-37, 39-40, 42-43, 46-47, 49, 51, 54, 56, 61-62, 66-69, 71, 73, 76, 81
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 12: 4, 9, 11, 15-17, 20-21, 26-27, 29, 34-36, 38-39, 41, 43, 46, 53-55, 66-69, 76, 78, 81
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 13: 1, 12, 16, 25-26, 29, 31, 35, 38-39, 41, 49-50, 53, 56-57, 60-62, 64-66, 70, 72-73
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 14: 1-4, 7-8, 10-11, 13-14, 18, 21-22, 27, 30-31, 35, 37, 40-41, 45, 47-49, 52-53, 57, 64, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 15: 2-3, 5, 7-9, 11, 13-16, 22-24, 28-29, 35-37, 40-41, 43-44, 49, 55, 60-61, 65, 67-69, 71, 75, 78, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 16: 1-3, 8-12, 15-17, 21-22, 24-25, 27, 30, 32-33, 35, 40, 43, 45, 51-52, 56, 58-60, 64, 66-68, 71-72, 75, 81, 83
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 17: 2-3, 15-16, 19, 22, 25-26, 31, 35, 38, 47-48, 51, 53, 56-59, 62-64, 67, 70-71, 76, 78-79
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 18: 2-3, 6-8, 11, 13-14, 16, 18, 26, 28, 35, 40, 43, 46-47, 50-53, 55, 58-59, 67, 69, 74, 77, 81-82
Error: (1431.1) FASTA-Reader: Warning: FASTA-Reader: Ignoring invalid residues at position(s): On line 19: 2, 5, 7, 11, 15, 17, 20, 22, 27, 34, 37, 40-41, 43-45, 47, 49, 52, 54-55

Any idea?

Thank you.

[joseph.fass]

I'm seeing this same thing within the Blast2GO CLI pipeline, but I've provided the blastx binary, and I'm blasting a nucleotide fasta (transcriptome) against SWISS-PROT, which is protein (swissprot.pal, etc) ... was your problem resolved by the answer below, or did it turn out to be something else?

[nikhilshinde0909]

Hi all

i am getting also same error with blastx with custom db but when i run with swissprot output is generating

here i am attaching command that i have used

blastx -query ~/Transdecoder_out/Rio/longest_orfs.pep -db ~/blastdb/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.pep -num_threads 8 -max_target_seqs 1 -outfmt 6 > Rio.outfmt6

Please someone guide

[GenoMax]

If you have peptide query sequences (looking at the file name) then you should be using blastp not blastx.

Answer 1

[Ram]

Hi,

Could you maybe give us the first few lines of the query file? I think it might be a protein and not a nucleotide query, or it might have other invalid characters. The output of `head contig2-query.fasta` would help a lot.

Thank you!

[arronar]

Yes you are right! It is a protein i should try with tblastn.

>gi|573913339|ref|XP_006644353.1| PREDICTED: probable pleiotropic drug resistance protein 2-like [Oryza brachyantha]
MDTAAEMQKVASLRRGGGGSSASMWWGADNGVFSRSRSSSMAEEDDEEALRWAALEKLPTYDRVRRAILPMEGGAAAGGGEGGAGGAGGEAGKRVVDVLSLGPQERRALLERLVRVAEDDNERFLLKLKERIDRVGIDIPTIEVRFEHLEAEAEVRVGNSGLPTVL

NSMTNKFEEAGNALGIVPNRKQTMPILHDVSGVIKPRRMTLLLGPPGSGKTTLLLALAGRLNKDLKFSGQVTYNGHQMDEFVP
QRTAAYISQHDLHIGEMTVRETLAFSARCQGVGTRFDMLTELSRREKAANIKPDADIDAFMKASAMEGQETNLITDYILKILG
LEICADTMVGDDMVRGISGGQRKRVTTGEMLVGPANALFMDEISTGLDSSTTFQIVKSLRQAIHILGGTAVISLLQPAPETYD
LFDDIILLSDGQIVYQGPREGVLEFFELMGFKCPERKGVADFLQEVTSKKDQKQYWMHHEKPYRYVPVKEFAGAFQSFHTGRS
IANELATPFDKSKSHPAALTTSRYGVSAMELLKANIDREFLLMKRNSFVYIFRTCQLMVVSLIAMTLFFRTKMHRDSVTDGVI
FMGALFFSVMMIMFNGLSELPLTIFKLPVFFKQRDLLFFPAWTYTIPTWILKTPMSFIEVGGFCFMSYYVIGFDPNVGRFFKQ
YLLMLAINQMAAAMFRFVGGAARNIIVANVFGSFMLLIFMVLGGFILVREKVKKWWIWGYWISPMMYAQNAISVNEFLGHSWV

[Ram]

You're welcome!