Help with PanPhlAn tutorial


I am following the PanPhlAn tutorial online and ran into an issue I can’t solve ( I am at step 4 (Build Bowtie2 indexes) but getting an error.

Error: Please provide input files, either (–fna & --ffn), (–gff & --fna), or --gff alone.

I am confused because the tutorial doesn’t mention -gff files or --ffa files.



there are currently two scripts with similar names. and

The first one comes from previous version where the user should provide fasta file and annotation files to generate the pangenome himself (using the USEARCH software).
The second script assume that you already have everything and only need. For the next step (mapping with, one only need to generate the Bowtie2 indexes from fna files.

Anyway, I confess this isn’t the best option anyway. We are currently working on a full pangenome database, providing fasta, annotation and Bowtie2 indexes, that should be available soon.

Thank you. I am quite new to bioinformatics so was struggling a bit. I have the new version of panphlan so can I create my own pangenome with fasta files and annotation files?


creating your own pangenome with fasta and annotation is actually part of the “old code” ( from PanPhlAn 1.2. For that you’ll need the USEARCH software for clustering (


Thank you. I have just realized that I was using the 1.2 version so I should be able to make my own pangenome. Also, the panphlan_erectale16_pangenome.csv export from ChocoPhlan looks different to the panphlan_39491_pangenome.csv in the tutorial. Is there a reason one shows the UniProtID and the other the locus tags instead?

Hi , I have been studying panphlan example

I download 14 fna and put them into reference_genomes/


when I run the follwing code I get an error,would you please give panphlan_39491_pangenome.csv file?

./panphlan/ \
	-c 39491 --i_fna reference_genomes/ -o . --verbose

(panphlan) [ckzhu@vm-login02 panphlan]$ ./panphlan/ \
> -c 39491 --i_fna reference_genomes/ -o . --verbose

 Error: Pangenome file (reference_genomes/panphlan_39491_pangenome.csv) not found

I also try old code

(panphlan) [ckzhu@vm-login02 panphlan]$ ./panphlan/ -c 39491 --i_fna reference_genomes/ -o . --verbose

PanPhlAn pangenome generation version
Python version: 3.7.3
System: linux
./panphlan/ -c 39491 --i_fna reference_genomes/ -o . --verbose

[I] Input genome FNA folder: /public/home/sample_lib/ckzhu/software/panphlan/reference_genomes/
[I] Species database name: 39491
[I] Identity threshold percentage: 95.0 %.
[I] Output folder: ./
[I] Temporary folder: TMP_panphlan_db/

STEP 1. Checking required software installations ...
[I] Bowtie2 is installed, version: 2.4.1, path: /public/home/sample_lib/ckzhu/miniconda3/envs/panphlan/bin/bowtie2
[I] Usearch v.7 is installed, version: v7.0.1090_i86linux32, path: /public/home/sample_lib/ckzhu/software/panphlan/usearch7

STEP 2. Prepare input gene and genome files ...
Traceback (most recent call last):
  File "./panphlan/", line 1341, in <module>
  File "./panphlan/", line 1294, in main
    path_genome_fna_files, path_gene_ffn_files = check_genomes(args['i_ffn'], args['i_fna'], VERBOSE)
  File "./panphlan/", line 1089, in check_genomes
    genefiles   = [f for f in os.listdir(ffn_folder) if fnmatch(f,'*.'+FFN)]
NotADirectoryError: [Errno 20] Not a directory: False

Is there something else I need to download?


I answered your question here :