Error in running Metaphlan 3 when using customized database

Microbial community profiling MetaPhlAn
1

Hello, @fbeghini and bioBakery team! I have added the genome of interest in the Chocophlan database using the instructions mentioned in here and here. I got the following error: of glen while using the new Chocophlan database.

Command:
metaphlan kneaddata_output/R1_kneaddata_paired_1.fastq, kneaddata_output/R2_kneaddata_paired_ 2.fastq --input_type fastq -o metaphlan_output/LW_NewDb.tsv -t rel_ab_w_read_stats --stat_q 0.2 --min_alignment_len 30 --nproc 64 --add_viruses --bowtie2db /home/samiahkanwar/Desktop/AKU_System/MetaPhlAn3/bowtie2db --add_viruses --bowtie2out metaphlan_output/LW_NewDB.bz2

Error:

> Traceback (most recent call last):
>   File "/home/samiahkanwar/anaconda3/envs/mpa/bin/metaphlan", line 8, in <module>
>     sys.exit(main())
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 1071, in main
>     tree, map_out = create_tree_and_add_reads( mpa_pkl, ignore_markers, pars, avg_read_length, markers2reads)
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 936, in create_tree_and_add_reads
>     tree = TaxTree( mpa_pkl, ignore_markers , unknown_calculation = unknown_calculation)
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 687, in __init__
>     add_lens(self.root)
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 679, in add_lens
>     lens.append( add_lens( c ) )
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 679, in add_lens
>     lens.append( add_lens( c ) )
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 679, in add_lens
>     lens.append( add_lens( c ) )
>   [Previous line repeated 4 more times]
>   File "/home/samiahkanwar/anaconda3/envs/mpa/lib/python3.7/site-packages/metaphlan/metaphlan.py", line 676, in add_lens
>     return node.glen
> AttributeError: 'TaxClade' object has no attribute 'glen'

The above command does create the bowtie2 output file, and when I used this file as input for Metaphlan, the command ran successfully and generated the relative abundance file.
I’m wondering why this is happening.

  1. Why is Metaphlan unable to generate an output file when using fastq files as input and a customized database?

Note: Metaphlan runs without errors when I use the default chocophlan database (MPA_v30_CHOCOPhlAn_201901) with fastq files as input .

  1. Can I relay on the results generated by this metaphlan metaphlan_output/LW_newDB.bz2 --nproc 5 --input_type bowtie2out -o metaphlan_output/LW_newDB.tsv -t rel_ab_w_read_stats --stat_q 0.2 --min_alignment_len 30 --add_viruses command or has it missed some important steps?
    Note: Bowtie2 output file contains the hits from the new marker sequence.