Trouble importing fasta reads

dac330 · June 12, 2017, 6:17pm

Hello:

I have run into some trouble trying to inspect my sequence assignments and alignments using mEGAN – notably, I have been unable to successfully import my fastA reads.

I’ve produced an assignment file that is tab-separated (blastn/.m8) using the program USEARCH for importing into MEGAN. At the same time, I have also tried to import the associated sequences file (fasta). Once the program completes the analysis, I click on a node to show read alignments, and I get an error message:

Error: Execute failed: java.io.IOException: Can’t display alignments, reads sequences appear to be missing from RMA file

The program does not appear to be recognizing the read sequences fasta file. I am unsure why, as I’ve modified my files to make sure they are in the proper format. I’ve also tried uploading SAM and human readable formats and still have the same error message. Finally, I’ve tried uploading my full fastA file (millions of sequences) vs. a truncated fastA, containing just the sequences that had been assigned/aligned. With each exercise, I still get the same error messages.

Is it possible to advise on this error?

Below is my program log.

Thank you in advance.

Program Notes:

Version MEGAN Community Edition (version 6.8.9, built 1 Jun 2017)
Copyright © 2017 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.

Opening startup files
Loading ncbi.map: 1,601,128
Loading ncbi.tre: 1,601,131
Induced tree has 1,601,131 of 1,601,131 nodes
Executing: show window=ImportBlast;
Executing: load mapFile=’/Users/Sterenn/Desktop/nucl_acc2tax-May2017.abin’ mapType=Accession cName=Taxonomy;
Opening file: /Users/Sterenn/Desktop/nucl_acc2tax-May2017.abin
Executing: import blastFile=’/Users/Sterenn/Desktop/nt_ublast_5’ fastaFile=’/Users/Sterenn/Desktop/output.fasta’ meganFile=’/Users/Sterenn/Desktop/nt_ublast_5.rma6’ useCompression=false format=BlastTab mode=BlastN maxMatches=100 minScore=50.0 maxExpected=0.01 minPercentIdentity=0.0 topPercent=10.0 minSupportPercent=0.01 minSupport=1 lcaAlgorithm=naive minPercentReadToCover=0.0 minComplexity=0.0 useIdentityFilter=false readAssignmentMode=readCount fNames=;
Classifications: Taxonomy
Parsing file: /Users/Sterenn/Desktop/nt_ublast_5
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:1874:2751’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:7948:2875’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:8252:2945’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:8934:2871’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:11292:2775’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:10953:2919’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:19412:2844’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:17751:2774’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:5245:3051’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:3451:3107’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:9796:3020’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:7825:3081’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:12231:3062’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:14483:3232’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:18289:3102’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:17710:3095’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:2492:3364’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:8583:3332’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:12213:3462’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:14088:3408’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:8363:3614’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:19310:3606’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:3745:3751’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:6415:3968’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:4703:3928’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:10519:3760’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:14606:3965’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:13909:3768’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:17606:3907’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:4052:3881’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:9467:4066’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:9681:4085’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:17916:4188’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:2988:4387’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:15281:4342’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:11698:4525’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:12072:4747’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:4475:4655’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:19385:4598’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:3005:4790’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:1853:4900’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:4157:4769’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:4446:4902’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:9224:4834’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:8457:4965’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:17199:4806’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:4682:5230’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:7605:5232’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:10212:5009’ in file: /Users/Sterenn/Desktop/output.fasta
WARNING: Failed to find read ‘HWI-D00653:88:H3J53BCXX:2:1101:14912:5161’ in file: /Users/Sterenn/Desktop/output.fasta
No further ‘failed to find read’ warnings…
Total reads: 220,640
Alignments: 613,419
Binning reads…
Using Naive LCA algorithm for binning: Taxonomy
Total reads: 220,640
With hits: 220,640
Alignments: 613,419
Assig. Taxonomy: 220,637
MinSupport set to: 22
Min-supp. changes: 2,275
Numb. Tax. classes: 1,016
Class. Taxonomy: 1,016
Induced tree has 1,724 of 1,601,131 nodes
Info: Command completed (61s): import blastFile=’/Users/Sterenn/Desktop/nt_ublast_5’ fastaFile=’/Users/Sterenn/Desktop/output.fasta’ meganFile=’/Users/Stere…
Induced tree has 1,724 of 1,601,131 nodes
Executing: show window=aligner;
Collecting data…
Can’t display alignments, reads sequences appear to be missing from RMA file
Command usage: show window=aligner; - Show alignment of reads to a specified reference sequence
Can’t display alignments, reads sequences appear to be missing from RMA file
Error: Execute failed: java.io.IOException: Can’t display alignments, reads sequences appear to be missing from RMA file

Daniel · July 7, 2017, 3:34pm

Please check that the following two conditions are met:

the names of the reads are exactly the same both in the alignment file and in the fasta file.
In both cases, the name must be a single word (the first word that appears on the fasta header line)
the reads must appear in exactly the same order in both the alignment file and the fasta file, because MEGAN streams both files simultaneously.

Please fix, if necessary. If this was not the cause of the problem, then please send me a small example of an alignment file and corresponding fasta file to take a look at.