New search on Megan

User Questions
1

Hi there,
I am new at Megan so please forgive me for some basic questions.
I have run blastn for align my files against ncbi database.
I obtained a output file that I would visualize in Megan. However, I loaded the blast output file in the “import from Blast” and then I try to run Megan6 but the results show me that 27 reads are “not Assigned” (out of 51Million No Hits).

image
image1719×868 4.1 KB

I am wondering to know what species are related to these reads, but I cannot find how to obtain this information.
I tried to put in the taxonomy menu the last “megan-nucl-map-Jul2020.db” file but the result is the same.
How can I proceed?
Thanks

2

Could you please show what outline MEGAN reported to the message window…

3

Hi, thank you for response me.
here is the the message window:

Version MEGAN Community Edition (version 6.20.18, built 15 Dec 2020)
Author(s) Daniel H. Huson
Copyright © 2020 Daniel H. Huson. This program comes with ABSOLUTELY NO WARRANTY.
This is free software, licensed under the terms of the GNU General Public License, Version 3.
Sources available at: https://github.com/husonlab/megan-ce
Java version: 14.0.1

Opening startup files
Loading ncbi.map: 2,259,889
Loading ncbi.tre: 2,259,893
Executing: show window=ImportBlast;
Executing: import blastFile=‘D:\sabrina_fungi’ meganFile=‘D:\sabrina_fungi-18.rma6’ useCompression=false format=BlastText mode=BlastN maxMatches=1 minScore=50.0 maxExpected=0.01 minPercentIdentity=0.0 topPercent=10.0 minSupportPercent=0.05 lcaAlgorithm=naive minPercentReadToCover=0.0 minPercentReferenceToCover=0.0 minComplexity=0.0 useIdentityFilter=false readAssignmentMode=readCount fNames=;
Classifications: Taxonomy
Annotating RMA6 file using EXTENDED mode
Parsing file: D:\sabrina_fungi
Total reads: 51,038,284
Alignments: 27
Initializing binning…
Using ‘Naive LCA’ algorithm for binning: Taxonomy
Binning reads…
Total reads: 51,038,284
With hits: 27
Alignments: 27
Assig. Taxonomy: 0
MinSupport set to: 25519
Numb. Tax. classes: 2
Class. Taxonomy: 2
Info: Command completed (5873s): import blastFile=‘D:\sabrina_fungi’ meganFile=‘D:\sabrina_fungi-18.rma6’ useCompression=false format=BlastText mode=BlastN …

4

You need to specify a mapping file. Please download this file: https://software-ab.informatik.uni-tuebingen.de/download/megan6/megan-nucl-Jul2020.db.zip
Unzip it and then supply it to Megan as mapping db file on the Taxonomy tab

Please let me know whether that fixes your problem

5

Hi Daniel,
I used the suggested files but the result is the same.
here is the message:
Opening startup files
Loading ncbi.map: 2,259,889
Loading ncbi.tre: 2,259,893
Executing: show window=ImportBlast;
Executing: use cViewer=EC state=false;
Executing: use cViewer=EGGNOG state=false;
Executing: use cViewer=GTDB state=false;
Executing: use cViewer=INTERPRO2GO state=false;
Executing: use cViewer=KEGG state=false;
Executing: use cViewer=SEED state=false;
Executing: load mapFile=‘D:\megan-nucl-Jul2020.db\megan-nucl-map-Jul2020.db’ mapType=MeganMapDB cName=Taxonomy;
Executing: use cViewer=Taxonomy state=true;
Executing: update;
Executing: set accessionMapMode=extended;
Executing: update;
Executing: import blastFile=‘D:\sabrina_fungi’ meganFile=‘D:\sabrina_fungi-1.rma6’ useCompression=false format=BlastText mode=BlastN maxMatches=1 minScore=50.0 maxExpected=0.01 minPercentIdentity=0.0 topPercent=10.0 minSupportPercent=0.05 lcaAlgorithm=naive minPercentReadToCover=0.0 minPercentReferenceToCover=0.0 minComplexity=0.0 useIdentityFilter=false readAssignmentMode=readCount fNames=;
Classifications: Taxonomy
Annotating RMA6 file using EXTENDED mode
Parsing file: D:\sabrina_fungi
Total reads: 51,038,284
Alignments: 27
Initializing binning…
Using ‘Naive LCA’ algorithm for binning: Taxonomy
Binning reads…
Total reads: 51,038,284
With hits: 27
Alignments: 27
Assig. Taxonomy: 27
MinSupport set to: 25519
Min-supp. changes: 7
Numb. Tax. classes: 2
Class. Taxonomy: 2
Info: Command completed (6137s): import blastFile=‘D:\sabrina_fungi’ meganFile=‘D:\sabrina_fungi-1.rma6’ useCompression=false format=BlastText mode=BlastN m…

and the plot:

image
image1781×863 4.13 KB

Can you suggest me further steps?
thanks

6

could you send me the first 1,000 or 10,000 lines of your blast file…

7

BLASTN 2.11.0+

Reference: Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb
Miller (2000), “A greedy algorithm for aligning DNA sequences”, J
Comput Biol 2000; 7(1-2):203-14.

Database: Internal transcribed spacer region (ITS) from Fungi type and
reference material
12,165 sequences; 7,153,958 total letters

Query= ST-E00127:1028:H73GKCCX2:6:1101:3153:1854 1:N:0:CAGCTA

Length=151

***** No hits found *****

Lambda K H
1.33 0.621 1.12

Gapped
Lambda K H
1.28 0.460 0.850

Effective search space used: 896804090

Query= ST-E00127:1028:H73GKCCX2:6:1101:3234:1854 1:N:0:CAGCTA

Length=151

***** No hits found *****

Lambda K H
1.33 0.621 1.12

Gapped
Lambda K H
1.28 0.460 0.850

Effective search space used: 896804090

Query= ST-E00127:1028:H73GKCCX2:6:1101:4148:1854 1:N:0:CAGCTA

Length=151

***** No hits found *****

Lambda K H
1.33 0.621 1.12

Gapped
Lambda K H
1.28 0.460 0.850

Effective search space used: 896804090

Query= ST-E00127:1028:H73GKCCX2:6:1101:4229:1854 1:N:0:CAGCTA

Length=151

***** No hits found *****

Lambda K H
1.33 0.621 1.12

Gapped
Lambda K H
1.28 0.460 0.850

Effective search space used: 896804090

#AFTER A LOT OF THIS “NO HITS” (I have searched the hits through sed command)

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 287 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 346

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 347 TCAAGCCTCGCT 358

NR_163324.1 Cladosporium entadae MFLUCC 17-0919 ITS region; from TYPE material
Length=488

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 269 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 328

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 329 TCAAGCCTCGCT 340

NR_158360.1 Cladosporium endophytica MFLUCC 17-0599 ITS region; from TYPE
material
Length=581

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 317 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 376

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 377 TCAAGCCTCGCT 388

NR_156582.1 Ramularia mali CBS 129581 ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_156581.1 Ramularia glennii CBS 129441 ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_156348.1 Cladosporium domesticum CPC 22307 ITS region; from TYPE material
Length=555

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 302 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 361

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 362 TCAAGCCTCGCT 373

NR_156347.1 Cladosporium coloradense CPC 22238 ITS region; from TYPE material
Length=625

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 377 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 436

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 437 TCAAGCCTCGCT 448

NR_154953.1 Ramularia vallisumbrosae CBS 272.38 ITS region; from TYPE material
Length=593

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 350 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 409

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 410 TCAAGCCTCGCT 421

NR_154951.1 Ramularia rubella CPC 25911 ITS region; from TYPE material
Length=569

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_154950.1 Ramularia osterici CPC 10750 ITS region; from TYPE material
Length=578

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 349 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 408

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 409 TCAAGCCTCGCT 420

NR_154949.1 Ramularia lamii var. lamii CBS 108970 ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_154948.1 Ramularia heraclei CBS 108969 ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_154944.1 Ramularia collo-cygni CBS 101180 ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 349 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 408

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 409 TCAAGCCTCGCT 420

NR_154943.1 Ramularia armoraciae CBS 241.90 ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_154942.1 Ramularia abscondita ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 347 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 406

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 407 TCAAGCCTCGCT 418

NR_154919.1 Ramularia hydrangeicola KACC 43597 ITS region; from TYPE material
Length=495

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 259 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 318

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 319 TCAAGCCTCGCT 330

NR_154917.1 Ramularia pusilla CBS 124973 ITS region; from TYPE material
Length=591

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 349 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 408

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 409 TCAAGCCTCGCT 420

NR_154911.1 Ramularia haroldporteri CPC 16296 ITS region; from TYPE material
Length=598

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 356 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 415

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 416 TCAAGCCTCGCT 427

NR_154901.1 Ramularia cynarae CPC 18426 ITS region; from TYPE material
Length=593

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 371 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 430

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 431 TCAAGCCTCGCT 442

NR_154899.1 Ramularia stellariicola ITS region; from TYPE material
Length=645

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 350 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 409

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 410 TCAAGCCTCGCT 421

NR_154898.1 Ramularia acroptili CBS 120252 ITS region; from TYPE material
Length=645

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 350 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 409

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 410 TCAAGCCTCGCT 421

NR_152307.1 Cladosporium chasmanthicola CPC 21300 ITS region; from TYPE material
Length=589

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 360 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 419

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 420 TCAAGCCTCGCT 431

NR_152296.1 Cladosporium pseudochalastosporoides ITS region; from TYPE material
Length=599

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 360 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 419

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 420 TCAAGCCTCGCT 431

NR_152295.1 Cladosporium penidielloides ITS region; from TYPE material
Length=599

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 365 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 424

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 425 TCAAGCCTCGCT 436

NR_152286.1 Cladosporium angustiherbarum ITS region; from TYPE material
Length=598

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 359 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 418

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 419 TCAAGCCTCGCT 430

NR_152308.1 Cladosporium welwitschiicola CPC 18648 ITS region; from TYPE
material
Length=535

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 300 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 359

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 360 TCAAGCCTCGCT 371

NR_145121.1 Ramularia eucalypti ITS region; from TYPE material
Length=588

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 348 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 407

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 408 TCAAGCCTCGCT 419

NR_145097.1 Ramularia proteae CBS 112161 ITS region; from TYPE material
Length=683

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 390 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 449

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 450 TCAAGCCTCGCT 461

NR_145125.1 Ramularia hydrangeae-macrophyllae CBS 122273 ITS region; from
TYPE material
Length=612

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 371 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 430

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 431 TCAAGCCTCGCT 442

NR_154947.1 Ramularia geranii CBS 160.24 ITS region; from TYPE material
Length=612

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 370 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 429

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 430 TCAAGCCTCGCT 441

NR_154946.1 Ramularia euonymicola CBS 113308 ITS region; from TYPE material
Length=480

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 258 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 317

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 318 TCAAGCCTCGCT 329

NR_154945.1 Ramularia digitalis-ambiguae CBS 297.37 ITS region; from TYPE
material
Length=592

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 368 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 427

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 428 TCAAGCCTCGCT 439

NR_154941.1 Ramularia citricola CPC 26192 ITS region; from TYPE material
Length=531

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 289 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 348

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 349 TCAAGCCTCGCT 360

NR_152300.1 Cladosporium aggregatocicatricatum ITS region; from TYPE material
Length=641

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 402 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 461

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 462 TCAAGCCTCGCT 473

NR_152299.1 Cladosporium rhusicola ITS region; from TYPE material
Length=639

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 400 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 459

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 460 TCAAGCCTCGCT 471

NR_152298.1 Cladosporium puyae CBS 274.80A ITS region; from TYPE material
Length=638

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 399 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 458

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 459 TCAAGCCTCGCT 470

NR_152294.1 Cladosporium aciculare ITS region; from TYPE material
Length=635

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 396 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 455

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 456 TCAAGCCTCGCT 467

NR_152293.1 Cladosporium parapenidielloides ITS region; from TYPE material
Length=590

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 360 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 419

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 420 TCAAGCCTCGCT 431

NR_152297.1 Cladosporium versiforme ITS region; from TYPE material
Length=536

Score = 134 bits (72), Expect = 4e-32
Identities = 72/72 (100%), Gaps = 0/72 (0%)
Strand=Plus/Plus

Query 1 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 297 ACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCAC 356

Query 61 TCAAGCCTCGCT 72
||||||||||||
Sbjct 357 TCAAGCCTCGCT 368

NR_152291.1 Cladosporium longicatenatum ITS region; from TYPE material
Length=635

AND SO ON…

8

Thank you, I checked some of the accessions, they appear to be present in megan-nucl-map-Jul2020.db

This is how you can check whether a given accession in present in a MEGAN mapping database:

Open the mapping database using the sqlite3:

sqlite3 megan-nucl-map-Jul2020.db

Query the accession (without the version suffixes):

sqlite> select * from mappings where Accession = ‘NR_158360’;
NR_158360|2259870
sqlite> select * from mappings where Accession = ‘NR_154953’;
NR_154953|215471
sqlite> select * from mappings where Accession = ‘NR_154948’;
NR_154948|1873284
…
.quit

So, that doesn’t seem to be the problem

To research this further, I need to load the data into MEGAN, so please give me access to the data as a file (not as pasted text, as that breaks the format)

Thanks

9

Hi Daniel, I have sent my file some days ago. have you received it? So you have some news?
Thanks for your help

10

Hi Daniel, this is a gentle reminder for you if you had some time to check the issues…
Thank you

11

I can’t find your file in my inbox- what was the subject line? (But that may just be me… my inbox is stupidly overstuffed…)

However, looking at the blastn hits that you posted, I see that the subject lines contain the taxon names, so you do not need to use the mapping database. Rather, make sure that “Parse Taxon Names” is selected, like this:

image
image2024×1624 411 KB

12

Thanks, Daniel! it should be
R: [MEGAN Community] [User Questions] New search on Megan
and the file is named Huson.
I am sending it again right now with a subject “File_Megan” from my Yale account.

13

Hi Daniel, I have tried to select only “Parse Taxon Names” but the result is the same.

Here is the message:
Version MEGAN Community Edition (version 6.21.0, built 18 Feb 2021)
Author(s) Daniel H. Huson
Copyright © 2020 Daniel H. Huson. This program comes with ABSOLUTELY NO WARRANTY.
This is free software, licensed under the terms of the GNU General Public License, Version 3.
Sources available at: https://github.com/husonlab/megan-ce
Java version: 14.0.1

Opening startup files
Loading ncbi.map: 2,302,807
Loading ncbi.tre: 2,302,811
Executing: show window=ImportBlast;
Executing: set idParsing=false cName=Taxonomy;
Executing: import blastFile=‘D:\sabrina_fungi’ meganFile=‘D:\sabrina_fungi-2.rma6’ useCompression=false format=BlastText mode=BlastN maxMatches=1 minScore=50.0 maxExpected=0.01 minPercentIdentity=0.0 topPercent=10.0 minSupportPercent=0.05 lcaAlgorithm=naive minPercentReadToCover=0.0 minPercentReferenceToCover=0.0 minComplexity=0.0 useIdentityFilter=false readAssignmentMode=readCount fNames=;
Classifications: Taxonomy
Annotating RMA6 file using EXTENDED mode
Parsing file: D:\sabrina_fungi
Total reads: 51,038,284
Alignments: 27
Initializing binning…
Using ‘Naive LCA’ algorithm for binning: Taxonomy
Binning reads…
Total reads: 51,038,284
With hits: 27
Alignments: 27
Assig. Taxonomy: 27
MinSupport set to: 25519
Min-supp. changes: 7
Numb. Tax. classes: 2
Class. Taxonomy: 2
Info: Command completed (5850s): import blastFile=‘D:\sabrina_fungi’ meganFile=‘D:\sabrina_fungi-2.rma6’ useCompression=false format=BlastText mode=BlastN m…

and the plot is

image
image1765×864 2.99 KB

14

I have no idea where those messages ending up… please send to daniel.huson@uni-tuebingen.de

15

Thanks for the file.

Using

grep -c “Query=” prova_fungi_100000000.blastn

the number of reads in the file is:
5,262,197

Using

grep -c “No hits found” prova_fungi_100000000.blastn

the number of reads that do not have any alignment is:
5,262,193

So, that means there are only 4 (= 5,262,197-5,262,193) reads in the file that have any alignment.

I ran the file through MEGAN anyway, and that is exactly what MEGAN reports:

Binning reads…
Total reads: 5,262,197
With hits: 4
Alignments: 400
Assig. Taxonomy: 4

There are exactly four reads that each have 100 alignments. All other reads have 0 hits.

(To see the four reads aligned to taxa, in MEGAN, during import you need to turn off the min-support filter, because, by default, only 4 reads out of 5 million reads aligning to any given taxon does not meet the min support threshold.)

So, MEGAN is working as intended, however, your reads are not aligning to anything and so they can not be binned.

image
image1920×2232 472 KB

16

Looking back at your previous posts, it appears that for the full file of 51 million reads, only 27 reads have alignments. So, you must have aligned your reads to an inappropriate database?