No assignment in MEGAN6 output

Question

[nisrinalulu]

Dear everyone,

I use DIAMOND and MEGAN6 CE for the first time for annotate my metagenomic data. Before I run my sequence to DIAMOND, first I contig my sequence using MEGAHIT and then blast it using blastx againts NCBI NR database using DIAMOND. I already have a .daa file and I converted it into the .ram file using daa2rma in MEGAN6 CE. I use database megan-map-Oct2019.db from MEGAN6 CE download page to annotate taxonomy, Interpro2go, SEED, and EggNOG.

Here is command line to make .rma file in MEGAN6:

daa2rma --in /home/husna/lulu/alignment/HB_result.daa --out /home/husna/lulu/rma-output/HB_result.rma --acc2taxa --acc2taxa-megan-map-Oct2019.db --acc2eggnog acc2eggnog-megan-map-Oct2019.db --acc2interpro2go --acc2interpro2go-megan-map-Oct2019.db --acc2seed --acc2seed-megan-map-Oct2019.db

And here is the output I get:

    Version   MEGAN Community Edition (version 6.12.3, built 14 Aug 2018)
    Copyright (C) 2018 Daniel H. Huson. This program comes with ABSOLUTELY NO WARRANTY.
    Functional classifications to use: EGGNOG, INTERPRO2GO, SEED
    Loading ncbi.map: 1,787,384
    Loading ncbi.tre: 1,787,388
    Caught:
    java.io.FileNotFoundException: --acc2taxa-megan-map-Oct2019.db (No such file or directory)
            at java.io.FileInputStream.open0(Native Method)
            at java.io.FileInputStream.open(FileInputStream.java:195)
            at java.io.FileInputStream.<init>(FileInputStream.java:138)
            at java.io.FileReader.<init>(FileReader.java:72)
            at jloda.util.FileInputIterator.<init>(FileInputIterator.java:98)
            at jloda.util.FileInputIterator.<init>(FileInputIterator.java:55)
            at megan.classification.data.Accession2IdMap.<init>(Accession2IdMap.java:45)
            at megan.classification.data.Accession2IdMapFactory.create(Accession2IdMapFactory.java:50)
            at megan.classification.IdMapper.loadMappingFile(IdMapper.java:148)
            at megan.tools.DAA2RMA6.run(DAA2RMA6.java:236)
            at megan.tools.DAA2RMA6.main(DAA2RMA6.java:64)
    Loading eggnog.map:    30,875
    Loading eggnog.tre:    30,986
    Caught:
  In DAA file:  /home/husna/lulu/alignment/HA_result_LR.daa
Output file:  /home/husna/lulu/rma-output/HA_result_2.rma
Classifications: Taxonomy,SEED,EGGNOG,INTERPRO2GO
Parsing file: /home/husna/lulu/alignment/HA_result_LR.daa
10% 20% 30% 40% 50% 60% 70% 80% 90% 100% (1207.7s)
Total reads:         1,032,501
Alignments:          6,360,907
100% (0.0s)
Binning reads: Initializing...
Initializing binning...
Using 'Naive LCA' algorithm for binning: Taxonomy
Using Best-Hit algorithm for binning: SEED
Using Best-Hit algorithm for binning: EGGNOG
Using Best-Hit algorithm for binning: INTERPRO2GO
Binning reads...
Binning reads: Analyzing alignments
Total reads:        1,032,501
With hits:           1,032,501
Alignments:          6,360,907
Assig. Taxonomy:             0
Assig. SEED:                 0
Assig. EGGNOG:               0
Assig. INTERPRO2GO:          0
MinSupport set to: 516
Binning reads: Applying min-support & disabled filter to Taxonomy...
Min-supp. changes:           0
Binning reads: Writing classification tables
Numb. Tax. classes:          1
Numb. SEED classes:          1
Numb. EGG. classes:          1
Numb. INT. classes:          1
Binning reads: Syncing
Using Best-Hit algorithm for binning: INTERPRO2GO
Binning reads...
Binning reads: Analyzing alignments
Total reads:        1,032,501
With hits:           1,032,501
Alignments:          6,360,907
Assig. Taxonomy:             0
Assig. SEED:                 0
Assig. EGGNOG:               0
Assig. INTERPRO2GO:          0
MinSupport set to: 516
Binning reads: Applying min-support & disabled filter to Taxonomy...
Min-supp. changes:           0
Binning reads: Writing classification tables
Numb. Tax. classes:          1
Numb. SEED classes:          1
Numb. EGG. classes:          1
Numb. INT. classes:          1
Binning reads: Syncing
Class. Taxonomy:             1
Class. SEED:                 1
Class. EGGNOG:               1
Class. INTERPRO2GO:          1
100% (1092.8s)
Total time:  2312s
Peak memory: 2.0 of 2.1G

I know there is wrong in my process but I don't know how to solved it. Please anyone give me guide to run MEGAN6.

Thank you so much for your help

Regards, Lulu

[Mensur Dlakic]

You seem to have too many double-dashes in this part:

--acc2taxa --acc2taxa-megan-map-Oct2019.db

Presumably, it should be:

--acc2taxa acc2taxa-megan-map-Oct2019.db

Your error as reported by the program:

FileNotFoundException: --acc2taxa-megan-map-Oct2019.db (No such file or directory)