gfflu is a Python CLI app to generate annotations of Influenza A virus (IAV) gene segment nucleotide sequences with
BLASTX and Miniprot using the same protein sequences as Influenza Virus Sequence Annotation Tool and
output a GFF3 file with the expected genetic features for each of the 8 IAV gene segments.
Table of Contents
Below is an example of typical usage with a FASTA nucleotide sequence file (Segment_4_HA.MH201222.fasta):
gfflu Segment_4_HA.MH201222.fastaProduces an output directory gfflu-outdir/ by default with the following files:
$ tree gfflu-outdir/
gfflu-outdir/
├── Segment_4_HA.MH201222.blastx.tsv
├── Segment_4_HA.MH201222.faa
├── Segment_4_HA.MH201222.gbk
├── Segment_4_HA.MH201222.gff
└── Segment_4_HA.MH201222.miniprot.gff
1 directory, 4 filesSpecify output directory with
-o /path/to/outdir
Help output:
Usage: gfflu [OPTIONS] FASTA
Annotate Influenza A virus sequences using Miniprot and BLASTX
The Miniprot GFF for a particular reference sequence gene segment will have multiple annotations for the same gene. This script will select the top scoring annotation for each gene and write out a new GFF file that can be used
with SnpEff.
â•â”€ Arguments ──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────╮
│ * fasta FILE Influenza virus nucleotide sequence FASTA file [default: None] [required] │
╰──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────╯
â•â”€ Options ────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────╮
│ --outdir -o PATH Output directory [default: gfflu-outdir] │
│ --force -f Overwrite existing files │
│ --prefix -p TEXT Output file prefix [default: None] │
│ --verbose -v │
│ --version -V Print 'gfflu version 0.0.2' and exit │
│ --install-completion Install completion for the current shell. │
│ --show-completion Show completion for the current shell, to copy it or customize the installation. │
│ --help Show this message and exit. │
╰──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────╯
gfflu version 0.0.2; Python 3.10.5 This is the recommended installation method.
conda install -c bioconda gfflupip install gffluThis install method assumes that you have BLAST+ and Miniprot
installed and on your$PATH.
Recommended to use conda to manage the environment from
the provided environment.yml file.
git clone https://github.com/CFIA-NCFAD/gfflu.git
cd gfflu
conda env create -f environment.yml
conda activate gfflugfflu outputs a SnpEff compatible GFF with the same features identified as the
Influenza Virus Sequence Annotation Tool.
Influenza Virus Sequence Annotation Tool output
>Feature MH201221
16 2295 gene
gene PB2
16 2295 CDS
product polymerase PB2
protein_id MH201221p1
gene PB2
INFO: Length: 2316 nucleotides
INFO: Segment: 1 (PB2)
INFO: Sequence completeness: protein 1 - complete; nucleotide - complete
INFO: This sequence (MH201221) contains following signature mutation(s) that might confer high virulence of the virus: (E627K)
INFO: Virus type: influenza A
NCBI Genbank GFF for MH201221.1
##gff-version 3
#!gff-spec-version 1.21
#!processor NCBI annotwriter
##sequence-region MH201221.1 1 2316
##species https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=11320
MH201221.1 Genbank region 1 2316 . + . ID=MH201221.1:1..2316;Dbxref=taxon:11320;Name=1;gbkey=Src;isolation-source=embyonated chicken eggs;mol_type=viral cRNA;note=laboratory-derived;segment=1;serotype=H1N1;strain=A/PR/8_RGCDC-4%2C6/34
MH201221.1 Genbank gene 16 2295 . + . ID=gene-PB2;Name=PB2;gbkey=Gene;gene=PB2;gene_biotype=protein_coding
MH201221.1 Genbank CDS 16 2295 . + 0 ID=cds-AVY92608.1;Parent=gene-PB2;Dbxref=NCBI_GP:AVY92608.1;Name=AVY92608.1;gbkey=CDS;gene=PB2;product=polymerase PB2;protein_id=AVY92608.1
gfflu GFF
##gff-version 3
##sequence-region MH201221 1 2295
MH201221 miniprot gene 16 2295 3747 + . ID=gene-PB2;Identity=0.9631;Name=PB2;Positive=0.9842;Rank=1;Target=PB2%7CCDS%7Cpolymerase_PB2%7CSeg1prot1A 1 759;gene=PB2;gene_biotype=protein_coding
MH201221 miniprot CDS 16 2295 3747 . 0 ID=cds-PB2;Identity=0.9631;Parent=gene-PB2;Rank=1;Target=PB2%7CCDS%7Cpolymerase_PB2%7CSeg1prot1A 1 759;gene=PB2;product=polymerase PB2
Influenza Virus Sequence Annotation Tool output
>Feature CY147460
13 2286 gene
gene PB1
13 2286 CDS
product polymerase PB1
protein_id CY147460p1
gene PB1
107 370 gene
gene PB1-F2
107 370 CDS
product PB1-F2 protein
protein_id CY147460p2
gene PB1-F2
INFO: Length: 2316 nucleotides
INFO: Segment: 2 (PB1)
INFO: Sequence completeness: protein 1 - complete; protein 2 - complete; nucleotide - complete
INFO: Virus type: influenza A
NCBI Genbank GFF for CY147460.1
##gff-version 3
#!gff-spec-version 1.21
#!processor NCBI annotwriter
##sequence-region CY147460.1 1 2316
##species https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=1343803
CY147460.1 Genbank region 1 2316 . + . ID=CY147460.1:1..2316;Dbxref=taxon:1343803;Name=2;collection-date=1934;country=Puerto Rico;gbkey=Src;lab-host=? + egg2 passage(s);mol_type=viral cRNA;nat-host=human;note=Strain PR8-LVD2 is phenotypically distinct from PR8 molecular clone;segment=2;serotype=H1N1;strain=A/Puerto Rico/8-LVD2/1934
CY147460.1 Genbank sequence_feature 1 2316 . + . ID=id-CY147460.1:1..2316;Dbxref=IRD:NIGSP_JY2_00027.PB1;gbkey=misc_feature
CY147460.1 Genbank gene 13 2286 . + . ID=gene-PB1;Name=PB1;gbkey=Gene;gene=PB1;gene_biotype=protein_coding
CY147460.1 Genbank CDS 13 2286 . + 0 ID=cds-AGQ47939.1;Parent=gene-PB1;Dbxref=NCBI_GP:AGQ47939.1;Name=AGQ47939.1;gbkey=CDS;gene=PB1;product=polymerase PB1;protein_id=AGQ47939.1
CY147460.1 Genbank gene 107 370 . + . ID=gene-PB1-F2;Name=PB1-F2;gbkey=Gene;gene=PB1-F2;gene_biotype=protein_coding
CY147460.1 Genbank CDS 107 370 . + 0 ID=cds-AGQ47940.1;Parent=gene-PB1-F2;Dbxref=NCBI_GP:AGQ47940.1;Name=AGQ47940.1;gbkey=CDS;gene=PB1-F2;product=PB1-F2 protein;protein_id=AGQ47940.1
gfflu GFF
##gff-version 3
##sequence-region CY147460 1 2286
CY147460 miniprot gene 13 2286 3892 + . ID=gene-PB1;Identity=0.9762;Name=PB1;Positive=0.9974;Rank=1;Target=PB1%7CCDS%7Cpolymerase_PB1%7Cseg2prot1B 1 757;gene=PB1;gene_biotype=protein_coding
CY147460 miniprot CDS 13 2286 3892 . 0 ID=cds-PB1;Identity=0.9762;Parent=gene-PB1;Rank=1;Target=PB1%7CCDS%7Cpolymerase_PB1%7Cseg2prot1B 1 757;gene=PB1;product=polymerase PB1
CY147460 feature gene 107 370 . + . ID=gene-PB1-F2;Target=PB1-F2%7CCDS%7CPB1-F2_protein%7Cseg2prot2M;gene=PB1-F2;gene_biotype=protein_coding
CY147460 feature CDS 107 370 . + 0 ID=cds-PB1-F2;Parent=gene-PB1-F2;Target=PB1-F2%7CCDS%7CPB1-F2_protein%7Cseg2prot2M;gene=PB1-F2;product=PB1-F2 protein
Influenza Virus Sequence Annotation Tool output
>Feature CY146806
13 2163 gene
gene PA
13 2163 CDS
product polymerase PA
protein_id CY146806p1
gene PA
13 772 gene
gene PA-X
13 582 CDS
584 772
product PA-X protein
protein_id CY146806p2
exception ribosomal slippage
gene PA-X
INFO: Length: 2208 nucleotides
INFO: Segment: 3 (PA)
INFO: Sequence completeness: protein 1 - complete; protein 2 - complete; nucleotide - complete
INFO: Virus type: influenza A
NCBI Genbank GFF for CY146806.1
##gff-version 3
#!gff-spec-version 1.21
#!processor NCBI annotwriter
##sequence-region CY146806.1 1 2208
##species https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=1346461
CY146806.1 Genbank region 1 2208 . + . ID=CY146806.1:1..2208;Dbxref=taxon:1346461;Name=3;country=USA: Texas;gbkey=Src;lab-host=? + egg2 passage(s);mol_type=viral cRNA;nat-host=human;segment=3;serotype=H3N2;strain=A/Texas/JY2/unknown
CY146806.1 Genbank sequence_feature 1 2208 . + . ID=id-CY146806.1:1..2208;Dbxref=IRD:NIGSP_JY2_00014.PA;gbkey=misc_feature
CY146806.1 Genbank gene 13 2163 . + . ID=gene-PA;Name=PA;gbkey=Gene;gene=PA;gene_biotype=protein_coding
CY146806.1 Genbank CDS 13 2163 . + 0 ID=cds-AGO00320.1;Parent=gene-PA;Dbxref=NCBI_GP:AGO00320.1;Name=AGO00320.1;gbkey=CDS;gene=PA;product=polymerase PA;protein_id=AGO00320.1
CY146806.1 Genbank gene 13 772 . + . ID=gene-PA-X;Name=PA-X;gbkey=Gene;gene=PA-X;gene_biotype=protein_coding
CY146806.1 Genbank CDS 13 582 . + 0 ID=cds-AGO00321.1;Parent=gene-PA-X;Dbxref=NCBI_GP:AGO00321.1;Name=AGO00321.1;exception=ribosomal slippage;gbkey=CDS;gene=PA-X;product=PA-X protein;protein_id=AGO00321.1
CY146806.1 Genbank CDS 584 772 . + 0 ID=cds-AGO00321.1;Parent=gene-PA-X;Dbxref=NCBI_GP:AGO00321.1;Name=AGO00321.1;exception=ribosomal slippage;gbkey=CDS;gene=PA-X;product=PA-X protein;protein_id=AGO00321.1
TODO: handle/add "exception=ribosomal slippage" to PA-X CDS
gfflu GFF
##gff-version 3
##sequence-region CY146806 1 2163
CY146806 miniprot gene 13 2163 3758 + . ID=gene-PA;Identity=0.9986;Name=PA;Positive=1.0000;Rank=1;Target=PA%7CCDS%7Cpolymerase_PA%7Cseg3prot 1 716;gene=PA;gene_biotype=protein_coding
CY146806 miniprot CDS 13 2163 3758 . 0 ID=cds-PA;Identity=0.9986;Parent=gene-PA;Rank=1;Target=PA%7CCDS%7Cpolymerase_PA%7Cseg3prot 1 716;gene=PA;product=polymerase PA
CY146806 miniprot gene 13 772 1301 + . Frameshift=1;ID=gene-PA-X;Identity=0.9987;Name=PA-X;Positive=0.9987;Rank=1;Target=PA-X%7CCDS%7CPA-X_protein%7Cseg3prot2C 1 252;gene=PA-X;gene_biotype=protein_coding
CY146806 miniprot CDS 13 772 1301 . 0 Frameshift=1;ID=cds-PA-X;Identity=0.9987;Parent=gene-PA-X;Rank=1;Target=PA-X%7CCDS%7CPA-X_protein%7Cseg3prot2C 1 252;gene=PA-X;product=PA-X protein
Influenza Virus Sequence Annotation Tool output
>Feature MH201222.1
21 1721 gene
gene HA
21 1721 CDS
product hemagglutinin
protein_id MH201222.1p1
function receptor binding and fusion protein
gene HA
21 71 sig_peptide
72 1052 mat_peptide
product HA1
1053 1718 mat_peptide
product HA2
INFO: Length: 1753 nucleotides
INFO: Segment: 4 (HA)
INFO: Sequence completeness: protein 1 - complete; nucleotide - complete
INFO: Serotype: H1
INFO: Virus type: influenza A
NCBI Genbank GFF for MH201222.1
##gff-version 3
#!gff-spec-version 1.21
#!processor NCBI annotwriter
##sequence-region MH201222.1 1 1753
##species https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=11320
MH201222.1 Genbank region 1 1753 . + . ID=MH201222.1:1..1753;Dbxref=taxon:11320;Name=4;gbkey=Src;isolation-source=embyonated chicken eggs;mol_type=viral cRNA;note=laboratory-derived;segment=4;serotype=H1N1;strain=A/PR/8_RGCDC-4%2C6/34
MH201222.1 Genbank gene 21 1721 . + . ID=gene-HA;Name=HA;gbkey=Gene;gene=HA;gene_biotype=protein_coding
MH201222.1 Genbank CDS 21 1721 . + 0 ID=cds-AVY92609.1;Parent=gene-HA;Dbxref=NCBI_GP:AVY92609.1;Name=AVY92609.1;gbkey=CDS;gene=HA;product=hemagglutinin;protein_id=AVY92609.1
MH201222.1 Genbank signal_peptide_region_of_CDS 21 71 . + . ID=id-AVY92609.1:1..17;Parent=cds-AVY92609.1;gbkey=Prot
MH201222.1 Genbank mature_protein_region_of_CDS 72 1052 . + . ID=id-AVY92609.1:18..344;Parent=cds-AVY92609.1;gbkey=Prot;product=HA1
MH201222.1 Genbank mature_protein_region_of_CDS 1053 1718 . + . ID=id-AVY92609.1:345..566;Parent=cds-AVY92609.1;gbkey=Prot;product=HA2
gfflu GFF
##gff-version 3
##sequence-region MH201222 1 1721
MH201222 miniprot gene 21 1721 2545 + . ID=gene-HA;Identity=0.8233;Name=HA;Positive=0.8993;Rank=1;Target=HA%7CCDS%7Chemagglutinin%7Cseg4protA 1 566;gene=HA;gene_biotype=protein_coding
MH201222 miniprot CDS 21 1721 2545 . 0 ID=cds-HA;Identity=0.8233;Parent=gene-HA;Rank=1;Target=HA%7CCDS%7Chemagglutinin%7Cseg4protA 1 566;gene=HA;product=hemagglutinin
MH201222 feature signal_peptide_region_of_CDS 21 71 . + . ID=signal_peptide-HA;Parent=cds-HA,gene-HA
MH201222 miniprot mature_protein_region_of_CDS 72 1052 1413 + 0 ID=mature_protein-HA;Identity=0.7737;Parent=cds-HA,gene-HA;Rank=1;Target=HA%7Cmature_protein_region_of_CDS%7CHA1%7Cseg4matureA2 1 327;product=HA1
MH201222 miniprot mature_protein_region_of_CDS 1053 1718 1109 + 0 ID=mature_protein-HA;Identity=0.9279;Parent=cds-HA,gene-HA;Rank=1;Target=HA%7Cmature_protein_region_of_CDS%7CHA2%7Cseg4matureA3 1 222;product=HA2
Influenza Virus Sequence Annotation Tool output
>Feature MH085254
44 1540 gene
gene NP
44 1540 CDS
product nucleocapsid protein
protein_id MH085254p1
gene NP
INFO: Length: 1561 nucleotides
INFO: Segment: 5 (NP)
INFO: Sequence completeness: protein 1 - complete; nucleotide - complete
INFO: Virus type: influenza A
NCBI Genbank GFF for MH085254.1
gfflu GFF
##gff-version 3
##sequence-region MH085254 1 1540
MH085254 miniprot gene 44 1540 2469 + . ID=gene-NP;Identity=0.9438;Name=NP;Positive=0.9819;Rank=1;Target=NP%7CCDS%7Cnucleocapsid_protein%7Cseg5prot 1 498;gene=NP;gene_biotype=protein_coding
MH085254 miniprot CDS 44 1540 2469 . 0 ID=cds-NP;Identity=0.9438;Parent=gene-NP;Rank=1;Target=NP%7CCDS%7Cnucleocapsid_protein%7Cseg5prot 1 498;gene=NP;product=nucleocapsid protein
>Feature EF190976
21 1385 gene
gene NA
21 1385 CDS
product neuraminidase
protein_id EF190976p1
gene NA
INFO: Length: 1413 nucleotides
INFO: Segment: 6 (NA)
INFO: Sequence completeness: protein 1 - complete; nucleotide - complete
INFO: Serotype: N1
INFO: Virus type: influenza A
gfflu GFF
##gff-version 3
##sequence-region EF190976 1 1385
EF190976 miniprot gene 21 1385 2231 + . ID=gene-NA;Identity=0.8681;Name=NA;Positive=0.9149;Rank=1;Target=NA%7CCDS%7Cneuraminidase%7Cseg6prot1A 1 470;gene=NA;gene_biotype=protein_coding
EF190976 miniprot CDS 21 1385 2231 . 0 ID=cds-NA;Identity=0.8681;Parent=gene-NA;Rank=1;Target=NA%7CCDS%7Cneuraminidase%7Cseg6prot1A 1 470;gene=NA;product=neuraminidase
Influenza Virus Sequence Annotation Tool output
>Feature MH085255
24 782 gene
gene M1
24 782 CDS
product matrix protein 1
protein_id MH085255p1
gene M1
24 1005 gene
gene M2
24 49 CDS
738 1005
product matrix protein 2
protein_id MH085255p2
gene M2
INFO: Length: 1023 nucleotides
INFO: Segment: 7 (MP)
INFO: Sequence completeness: protein 1 - complete; protein 2 - complete; nucleotide - complete
INFO: This sequence (MH085255) contains following signature mutation(s) that might confer amantadine resistance: (V27A) (S31N)
INFO: Virus type: influenza A
gfflu GFF
##gff-version 3
##sequence-region MH085255 1 1005
MH085255 miniprot gene 24 782 1238 + . ID=gene-M1;Identity=0.9683;Name=M1;Positive=0.9881;Rank=1;Target=M1%7CCDS%7Cmatrix_protein_1%7Cseg7prot1 1 252;gene=M1;gene_biotype=protein_coding
MH085255 miniprot CDS 24 782 1238 . 0 ID=cds-M1;Identity=0.9683;Parent=gene-M1;Rank=1;Target=M1%7CCDS%7Cmatrix_protein_1%7Cseg7prot1 1 252;gene=M1;product=matrix protein 1
MH085255 miniprot gene 24 1005 435 + . ID=gene-M2;Identity=0.8454;Name=M2;Positive=0.9072;Rank=1;Target=M2%7CCDS%7Cmatrix_protein_2%7Cseg7prot2A 1 97;gene=M2;gene_biotype=protein_coding
MH085255 miniprot CDS 24 49 41 + 0 ID=cds-M2;Identity=1.0000;Parent=gene-M2;Rank=1;Target=M2%7CCDS%7Cmatrix_protein_2%7Cseg7prot2A 1 8;gene=M2;product=matrix protein 2
MH085255 miniprot CDS 738 1005 394 . 1 ID=cds-M2;Identity=0.8295;Parent=gene-M2;Rank=1;Target=M2%7CCDS%7Cmatrix_protein_2%7Cseg7prot2A 9 97;gene=M2;product=matrix protein 2
Influenza Virus Sequence Annotation Tool output
>Feature MH085256
25 717 gene
gene NS1
25 717 CDS
product nonstructural protein 1
protein_id MH085256p1
gene NS1
25 862 gene
gene NEP
gene_syn NS2
25 54 CDS
527 862
product nuclear export protein
note nonstructural protein 2
protein_id MH085256p2
gene NEP
INFO: Length: 886 nucleotides
INFO: Segment: 8 (NS)
INFO: Sequence completeness: protein 1 - complete; protein 2 - complete; nucleotide - complete
INFO: Virus type: influenza A
gfflu GFF
##gff-version 3
##sequence-region MH085256 1 862
MH085256 miniprot gene 25 862 553 + . ID=gene-NS2;Identity=0.9174;Name=NS2;Positive=0.9339;Rank=1;Target=NS2%7CCDS%7Cnonstructural_protein_2%7Cseg8prot2A 1 121;gene=NS2;gene_biotype=protein_coding
MH085256 miniprot CDS 25 54 44 + 0 ID=cds-NS2;Identity=0.9000;Parent=gene-NS2;Rank=1;Target=NS2%7CCDS%7Cnonstructural_protein_2%7Cseg8prot2A 1 10;gene=NS2;product=nonstructural protein 2
MH085256 miniprot CDS 527 862 509 . 0 ID=cds-NS2;Identity=0.9189;Parent=gene-NS2;Rank=1;Target=NS2%7CCDS%7Cnonstructural_protein_2%7Cseg8prot2A 11 121;gene=NS2;product=nonstructural protein 2
MH085256 miniprot gene 25 717 1074 + . ID=gene-NS1;Identity=0.9130;Name=NS1;Positive=0.9565;Rank=1;Target=NS1%7CCDS%7Cnonstructural_protein_1%7Cseg8prot1J 1 230;gene=NS1;gene_biotype=protein_coding
MH085256 miniprot CDS 25 717 1074 . 0 ID=cds-NS1;Identity=0.9130;Parent=gene-NS1;Rank=1;Target=NS1%7CCDS%7Cnonstructural_protein_1%7Cseg8prot1J 1 230;gene=NS1;product=nonstructural protein 1
gfflu is distributed under the terms of the MIT license.