# ERROR : invalid value for fragment size ()

[jvollme]

i am having the same issue as #1 . I am analyzing a set of mags with the -metagenomic option. I do not quite understand why to use the "genomic" option here, because as the MAGs are binned from metagenomes, they do not have any larger scaffolds than the metagenomes themselves...

The protein-fastas are all correct. and the MAGS are mostly >90% complete (checkm2-estimation).

But one weird thing i noticed:
The error does NOT happen consistently. it may run perfectly well for the first 10 MAGS, and then fail with the abve mentioned error for all the rest. If i then redo this for all the failed mags, it works fine for the first five or so and THEN fails. and i can redo this indefinitley, again and again until (almost) all mags are analyzed (although the last 3 or so seem to keep failing now).

here one of the outputs:

nohup: ignoring input
+ perl mebs.pl -input /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3 -type metagenomic
# mebs.pl -input /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3 -type metagenomic -fdr 0.01 -comp 0

# Computing Mean Size Length (MSL) ...
# metabat2_gthaag_bin.0010_totalprots.faa MSL=246 MSLbin=250
# concoct_HAAG_biofilm_malegs1_bin.51_totalprots.faa MSL=298 MSLbin=300
# maxbin_HAAG_malegs2_bin.0003_totalprots.faa MSL=273 MSLbin=250

	sulfur	carbon	oxygen	iron	nitrogen	markers
vamb_HAAG_plant_bin.0001_totalprots.faa	4.868	5.877	-0.206	1.674	6.356	150.000*
metabat2_gthaag_bin.0010_totalprots.faa	5.067	5.913	1.061	2.789	6.909	138.000*
concoct_HAAG_biofilm_malegs1_bin.51_totalprots.faa	10.687*	13.385	4.332	6.180	12.204	155.000*
maxbin_HAAG_malegs2_bin.0003_totalprots.faaUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/maxbin_HAAG_malegs2_bin.0003_totalprots.faa.sulfur.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/maxbin_HAAG_malegs2_bin.0003_totalprots.faa.carbon.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/maxbin_HAAG_malegs2_bin.0003_totalprots.faa.oxygen.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/maxbin_HAAG_malegs2_bin.0003_totalprots.faa.iron.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/maxbin_HAAG_malegs2_bin.0003_totalprots.faa.nitrogen.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/maxbin_HAAG_malegs2_bin.0003_totalprots.faa.markers.score
	NA
vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faaUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faa.sulfur.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faa.carbon.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faa.oxygen.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faa.iron.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faa.nitrogen.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs2_bin.0035_totalprots.faa.markers.score
	NA
vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faaUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faa.sulfur.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faa.carbon.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faa.oxygen.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faa.iron.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faa.nitrogen.score
	NAUse of uninitialized value in concatenation (.) or string at mebs.pl line 287.
# ERROR : invalid value for fragment size ()
# ERROR: failed to generate /home/ww5070/projects/2025/geothermie/results/mebs/2_mags/proteins_failed3/vamb_HAAG_biofilm_malegs1_bin.0029_totalprots.faa.markers.score
	NA

[jvollme]

... also i noticed that the first MAG that gets a result line for sulfur- oxygen- etc cycling, is NOT the first one listed under "Computing Mean Size Length (MSL) ...".
So there seems to be some kind of shift in the order of the MAGs/Genomes being processed? Can i still assume that the results are all correctly associated with the correct genome-file?

[valdeanda]

Hello @jvollme
thanks a lot for the detailed report and logs!
So, I would suggest to use the 'genomic' option for MAGs. The reason for this is because it uses the full length of the protein family, rather than fragments of different sizes for the metagenomic option.

Even though MAGs come from metagenomes, they are assembled genomes (with full-length proteins).
In MEBS:

-type genomic uses the full length of the protein families during scoring (-size real), which is appropriate for MAGs/genomes.
-type metagenomic bins by fragment size to mimic short contigs/reads. That requires a valid fragment-size bin; if it can’t infer one, the variable is empty and you see: # ERROR : invalid value for fragment size ()
I would recommend to run mebs again with genomic mode and start fresh from a clean directory of protein FASTAs.

Let me know if switching to -type genomic resolves it for all your MAGs!
Thanks
Val