gfflu

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

• Usage
• Installation
  • Conda
  • PyPI
  • From Source
• Annotation
  • Segment 1
  • Segment 2
  • Segment 3
  • Segment 4
  • Segment 5
  • Segment 6
  • Segment 7
  • Segment 8
• License

Usage

Below is an example of typical usage with a FASTA nucleotide sequence file (Segment_4_HA.MH201222.fasta):

gfflu Segment_4_HA.MH201222.fasta

Produces 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 files

Specify 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.                                                                                                                                                                                                                           
                                                                                                                                                                                                                                        
╭─ Arguments ──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────╮
│ *    fasta      FILE  Influenza virus nucleotide sequence FASTA file [default: None] [required]                                                                                                                                      │
╰──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────╯
╭─ 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               

Installation

Conda

This is the recommended installation method.

conda install -c bioconda gfflu

PyPI

pip install gfflu

This install method assumes that you have BLAST+ and Miniprot
installed and on your $PATH.

From Source

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 gfflu

Annotation

gfflu outputs a SnpEff compatible GFF with the same features identified as the Influenza Virus Sequence Annotation Tool.

Segment 1

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

Segment 2

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

Segment 3

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

Segment 4

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

Segment 5

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

Segment 6

>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

Segment 7

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

Segment 8

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

License

gfflu is distributed under the terms of the MIT license.

References

• BLASTX
• Miniprot
• SnpEff
• Influenza Virus Sequence Annotation Tool
• GFF3