getting error in runing tblastn

Question

[inayat45shaikh]

Hello group,

I am trying to run tblastn of version 2.2.26+ for mapping peptides on eukaryotic genome, i am running the command

tblastn -query sequence.fa -out output.txt -subject ref_protein.fa -evalue 1e-5 -outfmt 7 -max_target_seqs 5 -best_hit_score_edge 0.05 -best_hit_overhang 0.25

but getting following error

Warning: lcl|Query_20167 contig20167: Warning: Could not calculate ungapped Karlin-Altschul parameters due to an invalid query sequence or its translation. Please verify the query sequence(s) and/or filtering options.

I used dustmasker also for filtering and masking and then run with masked genome, but still got the same error. I have also run by keeping filtering turn off with the following command

tblastn -query sequence.fa -out output.txt -F T -subject ref_protein.fa -evalue 1e-5 -outfmt 7 -max_target_seqs 5 -best_hit_score_edge 0.05 -best_hit_overhang 0.25

but got the following error

USAGE
  tblastn [-h] [-help] [-import_search_strategy filename]
    [-export_search_strategy filename] [-db database_name]
    [-dbsize num_letters] [-gilist filename] [-seqidlist filename]
    [-negative_gilist filename] [-entrez_query entrez_query]
    [-db_soft_mask filtering_algorithm] [-db_hard_mask filtering_algorithm]
    [-subject subject_input_file] [-subject_loc range] [-query input_file]
    [-out output_file] [-evalue evalue] [-word_size int_value]
    [-gapopen open_penalty] [-gapextend extend_penalty]
    [-xdrop_ungap float_value] [-xdrop_gap float_value]
    [-xdrop_gap_final float_value] [-searchsp int_value]
    [-max_hsps_per_subject int_value] [-db_gencode int_value]
    [-frame_shift_penalty frameshift] [-ungapped] [-max_intron_length length]
    [-seg SEG_options] [-soft_masking soft_masking] [-matrix matrix_name]
    [-threshold float_value] [-culling_limit int_value]
    [-best_hit_overhang float_value] [-best_hit_score_edge float_value]
    [-window_size int_value] [-lcase_masking] [-query_loc range]
    [-parse_deflines] [-outfmt format] [-show_gis]
    [-num_descriptions int_value] [-num_alignments int_value] [-html]
    [-max_target_seqs num_sequences] [-num_threads int_value] [-remote]
    [-comp_based_stats compo] [-use_sw_tback] [-in_pssm psi_chkpt_file]
    [-version]

DESCRIPTION
   Protein Query-Translated Subject BLAST 2.2.26+

Use '-help' to print detailed descriptions of command line arguments
========================================================================
Error: (CArgException::eInvalidArg) Unknown argument: "F"
Error: (CArgException::eInvalidArg) Application's initialization failed

Can anyone give advise on this?

Thanks,

[Pierre Lindenbaum]

as you can see, there is no '-F' option...

[Ram]

Guessing from your file naming, the query is nucl and the DB is protein. You might wanna try swapping the query and subject if that's the case, or use BLASTX/ explore other options.

Answer 1

[Prakki Rama]

The option -F for masking is only in the legacy blast for which one has to run blastall (which is different from blast+). Check the table in this page, a comparison between the masking options of blastall and blast+.

Moreover, as above Ram RS stated, your query should be protein and subject should be nucleotide sequences. If it is other way around, BLASTX is used to fulfill the purpose.

~Prakki Rama.

Answer 2

[Ginsea Chen]

You should add -soft_masking true option, then the error was displayed.