rcedgar / muscle Goto Github PK

Can you help out ? I just tried to execute Muscle on a fresh MacOS installation (version 12.1; dev tools enabled).

MBP-de-Nicolas:~ nicolas$ chmod +x /Users/nicolas/Desktop/muscle
MBP-de-Nicolas:~ nicolas$ /Users/nicolas/Desktop/muscle
dyld[995]: Library not loaded: /usr/local/opt/gcc/lib/gcc/11/libgomp.1.dylib
  Referenced from: /Users/nicolas/Desktop/muscle
  Reason: tried: '/usr/local/opt/gcc/lib/gcc/11/libgomp.1.dylib' (no such file), '/usr/local/lib/libgomp.1.dylib' (no such file), '/usr/lib/libgomp.1.dylib' (no such file)
Abort trap: 6

muscle -in combined.fasta -out align.fasta

MUSCLE v3.8.1551 by Robert C. Edgar

http://www.drive5.com/muscle
This software is donated to the public domain.
Please cite: Edgar, R.C. Nucleic Acids Res 32(5), 1792-97.

combined 74665 seqs, lengths min 38, max 11532, avg 307
Segmentation fault

Can you help me out with this?

dyld: Symbol not found: __ZNKSt7__cxx1112basic_stringIcSt11char_traitsIcESaIcEE4findEPKcmm
Referenced from: /Users/wang/src/BLCA/muscle
Expected in: /usr/local/lib/libstdc++.6.dylib
in /Users/wang/src/BLCA/muscle
Abort trap: 6

anyone knows how fix it? thank you

Hi,
I upgraded the Debian package to muscle 5.1. It builds nicely for Intel architectures. However, Debian builds for several other architectures and this was successful for version 3.8.1551. The autobuilders logs can be seen here. Looking for instance at arm64 log you can find at the end the failure:

g++ -Wdate-time -D_FORTIFY_SOURCE=2 -DNDEBUG -pthread -g -O2 -ffile-prefix-map=/<<PKGBUILDDIR>>=. -fstack-protector-strong -Wformat -Werror=format-security -O3 -fopenmp -ffast-math -c -o Linux/transaln.o transaln.cpp
In file included from testlog.cpp:2:
timing.h: In function ‘void cmd_testlog()’:
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
timing.h:26:9: error: impossible constraint in ‘asm’
   26 |         __asm__ __volatile__ ("rdtsc" : "=a" (lo), "=d" (hi));
      |         ^~~~~~~
make[2]: *** [Makefile:53: Linux/testlog.o] Error 1

This assembly is for x86 based system. On arm64 you would want something like this. It would be great if muscle would be portable also to other architectures.

Kind regards, Andreas.

ScoreType.h and FileBuffer.h are referenced in the code but not included with the release. They seem to come from amap, which is not listed as a dependency.

This is the first barrier that anybody wishing to build the code will run into.

This is very minor: Using the old syntax muscle -in input.fasta -out output.aln is not compatible anymore with version 5, but the error message does not point in the right direction. Instead of

Invalid command line
Unknown option in

It may be helpful to show something like

The option `--in` is no longer supported. Please use `--align` instead.

Hello Robert,

Any plans for M1/ARM structure support?

Thanks,

Jianshu

muscle -maketree -in MSA_RESULT.afa -out re.phy

Invalid command line
Unknown option maketree

muscle version-
muscle 5.0.1428_win64

Hello! I downloaded the latest linux binary but cannot get it to run. The steps I used are as follows:

chmod +x muscle
./muscle
zsh: exec format error:

The result is the same using the absolute path to the binary.

Hi @rcedgar,
thanks a ton for your efforts to provide useful tools to the community!

I was interested in switching from MAFFT to MUSCLE5 for large alignments of virus genomes.
There is feature in MAFFT (option --adjustdirection) which is able to reverse complement some sequences (quite useful because some similar virus genomes may have been deposited in different orientations in NCBI nuccore for example).
Do you think it could be implemented as well in MUSCLE5?

Best regards,

Thomas

This is a bug in the current code, I hope to post a fix in the next few days. Work-around is to write a script in python or similar -- it's pretty simple to read through the EFA file one line at a time. If the first character in the line is '<', start writing to a new file where the filename is the rest of this line (i.e. with '<' deleted).

muscle 5.0.1428_linux64 791Gb RAM, 24 cores
(C) Copyright 2004-2021 Robert C. Edgar.
https://drive5.com

Hello Robert,

I have the following error when running muscle. There supper5 one is good.

muscle -align ASVs1.fa -output aln.afa -threads 24
Elapsed time 01:27:16
Max memory 0.0b

---Fatal error---
mpcflat.cpp(170) assert failed: PairCount == PairCount2
5140.18user 87.93system 1:27:24elapsed 99%CPU (0avgtext+0avgdata 194959560maxresident)k
62000inputs+24outputs (11major+41593028minor)pagefaults 0swaps

Thanks,

Jianshu

Hi, I have successfully run Muscle 5.1.win64 on Windows once before. But this time, all files (including the one which succeeded before) failed to run and shows like:
---Fatal Error---
Cannot open xxx.fas , errno=2 No such file or directory

And I have checked the .fas or .fasta files. They all have right informations and can be open by other Editor softwares like BioEdit.

Can anyone help me?Thanks a lot.

Muscle &File address:
C:\Users\USER\Desktop\software\desktop\Muscle\muscle-5.1\muscle5.1.win64.exe

Code:
C:\Users\USER\Desktop\software\desktop\Muscle\muscle-5.1\muscle5.1.win64.exe -align ES_Mitos_match_unPaired_73_cob.fas -output test.fasta

.fas file is like:

SDHF_76M_cob
ATGAATAAATCTATACGAACTTATCACCCATTATTTAAAATTGCCAATAATGCTTAATTGAT????????????????????????????????????????GATCATTATTAGGAATTTGTTTAGTAAAACAAATCCTAACAGGAATATTYTTAGCCATACATTATAGCCCCAATATTKAACAAGCATTTTCTAGAGTAGCAMATATTTGTCGAGACGTAAATWATGGRTGACTACTTCGAGTCTTGCATGCCAATGGAGCATCTGTATTTTTTCTT????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????CTTGAGGACAAATATCTTTTTCTAGGGTGCCGGTGTCATAGTTTTTTGGAGCAGCCGTTCCATATTTAGGTACTGATCTAGTACAATGAGTATGAGGKGGCTTCGCTGTAGACAAYGCYACAYTAAACCGATTCTTTACTTTACATTTTTTAYTACCATTTATCATTACWGCACTTGTATTAATWCACTTAKTATTTCTTCACCAAACAGGATCAAAYAAMCCCATAGSAATAAACAGAAACATTGATAAAATTCCCTTCCATCCATACTTCACAACAAAAGACATTGTAGGRTTYATTATTATAWTTATAWTATTAKSCWTTTTATCACTCAAAGAACCATATATTCTTGGAGATCCTGACAATTTCACACCAGCTAATCCCTTAGTTACTCCTGTTCACATTCAACCTGAATGATATTTTCTATTTGCATATGCAATTTTACGATCAATTCCTAAYAAATTAGGRGGAGTAATCGCYYTAGTAATATCAATTGCYATCTTATTTATTATACCATTATATAAATCAAATTTTCGAAGAATACAATTTTACCCAATTAATCAAGCATACTTCTGAACAATAACAAATACTGTAATTTTATTAACATGAATTGGAGCACGACCAGTAGAAGAACCTTATATCATCACAGGCCAATTATTAACTACAATTTATTTTAGATATTATCTTATAATTCCTTTTATTACCAAAACATGAGAAACATAGACTTATG
SDHF_76R_cob
ATGAATAAAYCTATACGAACTCATCAYCCATTATTTAAAATTGCTAATAATGATTTAKTTGACCTTCCAGCCCCTTCAAATATTACAACCTGATGAAATTTTGAATCACTGTTAGGAATCTGCTTAATAATACAAATTTTAACGGGAATATTTCTAGCTATACATTATAGCCCAAACATTKAGCAAGCGTTTTCTAGAGTAACACACATTTGTCGAG???????????????????????????????????????????????????????????????????????????????TGGTCGASGAKWWYRSWAKRCRWSAWTMAAATTAATACATACCTGATTTGTAGGTGTAATAATTTTATTCATCACTATAGCAACAGCTTTTGTAGGGTATGTCTTACCTTGAGGACAAATATCTTTTTGAGGGGCCACTGTAATTACTAACCTTTTA?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????CTGTTTCTTCACYAWMYARSATCAAAYAACCCCTTTAGGAATAAATAGAAACATCGACAAAATCCCCTTCCATCCATATTTCACAACAAAAGACATTGTTGGATTCATCATTATATTTATAATATTATCTATTTTATCACTAAAAGAACCTTATATTCTAGGAGAYCCCGACAATTTCACACCAGCYAATCCTTTAGTTACYCCYATYCACATTCAACCTGAATGATATTTTCTATTTGCATACGCAATTTTACGATCAATTCCTAATAAATTAGGGGGAGTAATAGCCTTAGTGATATCAATTGCCATCTTATTTATTATACCATTATATAAATCAAATTTCCGAAGAATTCAATYCTACCCGATTAATCAAATATACTTCTGAATAATAGTAAACACTGTAATCTTATTAACATGAATTGGAGCACGACCAGTAGNNNNNNNNNNNTACAGGTCAATTATTAACTACASTMTATKTCAGATACTACMTWATWATTYCYTCAATTACTAAAATATGAGAAAATCTTAYCAAAT
SDHF_76S_cob
ATGAATAAATCTATACGAAYTWATCATCCATTATTTAAAATCGCCAATAATGCTTTAATTGATCTTCCGATCTCGAATAATTGAACATGATGAAACTTTGGATCATTATTAGGAATTTGTTTAGTAATACAAATCCTAACCGGAATATTCTTAGCCATACATTATAGACCTAATATCGAACAAGCATTTTCTAGAGTAGCACACATTTGTCGAGACGTAAATTATGGATGACTACTACGAACCTTACATGCTAATGGAGCMTCTATATTTTTTCTTTGTATTTACTTACA??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????CRRYKGSATCAAGATAACCCTTTAGGAATAAATAGAAACATCGACAAAATCCCCTTYCAYCCATATTWCACAACAAAAGAYATTGTTGGATTCATYATTATATTYATAVTAYTRTCHATTTTATCACTMAAAGAACCNNNNTTATATTCTAGGAGAYCCYGACAATTTTACACCGGCTAATCCTTTAGTTACCCCTGTTCACATTCAACCTGAATGGTACTTCCTATTTGCATATGCAATTTTACGATCAATTCCTAACAAATTAGGAGGAGTAATCGCCTTAGCAATATCAATTGCTATTCTATTTATCATACCATTACATAAATCAAATTTCCGAAGAATTCAATTTTACCCCAATT???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????
SDHF_76Z_cob
?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????ATAGTGTAAGACATCATTCAAATTAATACAGACCTGATTTGTAGGTGTAATAATTTTATTCATCACTATAGCAACAGCTTTTGTAGGATATGTCTTACCTTGAGGACAAATATCTTTTTGAGGGGCCACTGTAATTACTAACCTTTTATC???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????AATTCTCCATGGGAACGTACTTATATAATAATTCCCTCAATTACTAAAATATGAGAAAATCTTACCAAAT

muscle 5.0.1428_win64 34.1Gb RAM, 16 cores
(C) Copyright 2004-2021 Robert C. Edgar.
https://drive5.com

00:00 5.9Mb 0.037% Calc posteriors

D:\MyApp\bin\muscle.exe -align .\all.fasta -output all2.fasta
Elapsed time 00:04
Max memory 5.9Mb

---Fatal error---
myalloc64(4, 1.1e+09) failed

The default Makefile uses ccache, which is nonstandard and rarely installed on most modern systems. This is the first problem that someone who wishes to build the code will encounter Makefile_osx does not have this issue, although it does not build for me on up-to-date OSX with the standard clang-based Apple developer tools installed. That's a different issue, though.

There are many lines of rules for building cpp object, all of which can be replaced by the simple standard rule:

%.o: %.cpp $(DEPS)
$(CPP) -c -o $@ $&lt; $(CPPFLAGS)
muscle5: $(OBJS)
$(LNK) -o muscle5 $(LNKOPTS) $(OBJS)
if one does the standard practice of making object files in-place.

muscle 5.1.linux64 [12f0e2] 32.8Gb RAM, 16 cores
Built Jan 13 2022 23:17:13
(C) Copyright 2004-2021 Robert C. Edgar.
https://drive5.com

Input: 74665 seqs, avg length 308, max 11532

WARNING: >1k sequences, may be slow or use excessive memory, consider using -super5

Killed

i want to use it in .net core, use the main language is C#, but i want to run it in liunx, any one who can help me? thank you!

Hi I got the following error message when I use the CRISPRCasFinder, which includes MUSCLE as an integrated software. How can I fix this issue?

Fixed in rce-dev at time of writing (1/11/2022), will be committed to main in the next few days.
Work-around is to delete gaps in the input sequences.

openmp is from GCC and pthread are part of glibc. The makefile specifies -static for link, but the resulting binaries will not be fully static; I believe this is true for the distributed binaries, making it a problem that binary users will find a problem with.

/usr/lib/gcc/x86_64-pc-linux-gnu/11.1.0/../../../../x86_64-pc-linux-gnu/bin/ld: /usr/lib/gcc/x86_64-pc-linux-gnu/11.1.0/libgomp.a(oacc-profiling.o): in function `goacc_profiling_initialize':
/var/tmp/portage/sys-devel/gcc-11.1.0-r1/work/gcc-11.1.0/libgomp/oacc-profiling.c:137: warning: Using 'dlopen' in statically linked applications requires at runtime the shared libraries from the glibc version used for linking

The simple thing is to remove -static from the Makefile link. There are ways to build fully static for distribution, but they are more complicated than reflected in the makefile.

Hello,
I am trying to align my protein sequences and it runs well for a while but then it throws this error:

Floating point exception (core dumped)
Floating point exception (core dumped)
Floating point exception (core dumped)
etc.

Does anyone know why/what this means? Thanks!

I believe this is fixed in the latest main branch by adding this to the start of gitver.bash:

if [ ! -d ../.git ] ; then
  if [ ! -f gitver.txt ] ; then
    echo "0" > gitver.txt
  fi
  echo "Repo not found, git hash set to zero"
  exit 0
fi

I was using muscle5 for alignment of around 10k sequences, I am getting the following error:

muscle 5.1.linux64 [12f0e2]  528Gb RAM, 56 cores
Built Jan 13 2022 23:17:13
(C) Copyright 2004-2021 Robert C. Edgar.
https://drive5.com

Input: 9988 seqs, avg length 745, max 3209


WARNING: >1k sequences, may be slow or use excessive memory, consider using -super5

00:40 4.9Gb  CPU has 56 cores, running 36 threads
double free or corruption (!prev)eriors

Hello,
I'm sorry, but I fail to to compile Muscle 5.1 on Linux with GCC 11.2.1:

This is for Git snaphsot:

$ make
bash ./gitver.bash
"7630cd"
mkdir -p Linux/
g++  -DNDEBUG -pthread  -O3 -fopenmp -ffast-math -c -o Linux/addconfseq.o addconfseq.cpp
g++  -DNDEBUG -pthread  -O3 -fopenmp -ffast-math -c -o Linux/align.o align.cpp
...
g++  -DNDEBUG -pthread  -O3 -fopenmp -ffast-math -c -o Linux/usage.o usage.cpp
g++  -DNDEBUG -pthread  -O3 -fopenmp -ffast-math -c -o Linux/usorter.o usorter.cpp
g++  -O3 -fopenmp -pthread -lpthread -static Linux/addconfseq.o Linux/align.o Linux/alignpairflat.o Linux/allocflat.o Linux/alnalnsflat.o Linux/alnmsasflat.o Linux/alnmsasflat3.o Linux/alpha.o Linux/alpha3.o Linux/assertsameseqs.o Linux/buildposterior3flat.o Linux/buildpostflat.o Linux/bwdflat3.o Linux/calcalnflat.o Linux/calcalnscoreflat.o Linux/calcalnscoresparse.o Linux/calcposteriorflat.o Linux/colscoreefa.o Linux/consflat.o Linux/conspairflat.o Linux/defaulthmmparams.o Linux/derep.o Linux/diagbox.o Linux/disperse.o Linux/dividetree.o Linux/eacluster.o Linux/eadistmx.o Linux/eadistmxmsas.o Linux/eesort.o Linux/efabestcols.o Linux/efabestconf.o Linux/efaexplode.o Linux/efastats.o Linux/ensemble.o Linux/fasta.o Linux/fasta2.o Linux/fa2efa.o Linux/fwdflat3.o Linux/getconsseq.o Linux/getpairs.o Linux/getpostpairsalignedflat.o Linux/globalinputms.o Linux/guidetreejoinorder.o Linux/heatmapcolors.o Linux/help.o Linux/hmmdump.o Linux/hmmparams.o Linux/jalview.o Linux/jointrees.o Linux/letterconf.o Linux/letterconfhtml.o Linux/logaln.o Linux/logdistmx.o Linux/logmx.o Linux/main.o Linux/make_a2m.o Linux/maxcc.o Linux/mpcflat.o Linux/msa.o Linux/msastats.o Linux/msa2.o Linux/multisequence.o Linux/mysparsemx.o Linux/myutils.o Linux/pairhmm.o Linux/permutetree.o Linux/perturbhmm.o Linux/pprog.o Linux/pprogt.o Linux/pprog2.o Linux/probcons.o Linux/progalnflat.o Linux/project.o Linux/qscore.o Linux/qscoreefa.o Linux/qscorer.o Linux/qscore2.o Linux/quarts.o Linux/randomchaintree.o Linux/refineflat.o Linux/relabel.o Linux/relaxflat.o Linux/resample.o Linux/seb8.o Linux/seq.o Linux/sequence.o Linux/setprobconsparams.o Linux/stripgappycols.o Linux/stripgappyrows.o Linux/super4.o Linux/super5.o Linux/testfb.o Linux/testlog.o Linux/testscoretype.o Linux/textfile.o Linux/totalprobflat.o Linux/tracebackflat.o Linux/transaln.o Linux/transq.o Linux/tree.o Linux/treefromfile.o Linux/treeperm.o Linux/treesplitter.o Linux/treesubsetnodes.o Linux/treetofile.o Linux/tree2.o Linux/tree4.o Linux/trimtoref.o Linux/trimtorefefa.o Linux/uclust.o Linux/upgma5.o Linux/usage.o Linux/usorter.o -o Linux/muscle
/usr/lib64/gcc/x86_64-suse-linux/11/../../../../x86_64-suse-linux/bin/ld: cannot find -lm
/usr/lib64/gcc/x86_64-suse-linux/11/../../../../x86_64-suse-linux/bin/ld: cannot find -lc
collect2: error: ld returned 1 exit status
make: *** [Makefile:41: Linux/muscle] Error 1

Compiling released version ends with same error, just the beginning is different:

$ make
bash ./gitver.bash
fatal: not a git repository (or any parent up to mount point /)
Stopping at filesystem boundary (GIT_DISCOVERY_ACROSS_FILESYSTEM not set).
""
mkdir -p Linux/
g++  -DNDEBUG -pthread  -O3 -fopenmp -ffast-math -c -o Linux/addconfseq.o addconfseq.cpp
g++  -DNDEBUG -pthread  -O3 -fopenmp -ffast-math -c -o Linux/align.o align.cpp
...

It looks similar like an error I already got (discussed in #4), and looks bit low-level, but I'm sure I have all compilation tools correctly installed. I might be missing some dependency (although I think I checked everything), but I fail to find out which one it could be... When I succeed, I'll create package RPM for openSUSE Linux.

Trying to compile v5.0.1428:

$ make -C src/ CC=gcc CPP=g++ LNK=g++
...
g++ -fopenmp -ffast-math -msse -mfpmath=sse -O3 -DNDEBUG -c -o o/msa.o msa.cpp
g++ -fopenmp -ffast-math -msse -mfpmath=sse -O3 -DNDEBUG -c -o o/msa2.o msa2.cpp
g++ -fopenmp -ffast-math -msse -mfpmath=sse -O3 -DNDEBUG -c -o o/myutils.o myutils.cpp
myutils.cpp:2058:10: fatal error: ../ver/counter.h: No such file or directory
 2058 | #include "../ver/counter.h"
      |          ^~~~~~~~~~~~~~~~~~
compilation terminated.
make: *** [Makefile:381: o/myutils.o] Error 1

Dispersion calculation gives me two values, I dont which is the one that is mentioned on the website (and if <0.05 then alignment is likely fine):
@disperse file=ensemble_mafft.efa D_LP=0.005485 D_Cols=1

What is D_LP and D_Cols?

Hello,

I use MUSCLE quite often, but I think the speed of the program suffers a bit from the multi-threading algorithm.

It is quite common for me that after a some time (e.g. 50% of the total time needed for one pass of the Consistency step) more than half of the threads will have finished and it takes a long time for the last 2-3 threads to finish the work. I attach a btop screenshot with the pattern of core usage of a muscle (5.1.linux64) run:

The two large large "blocks" of activity on the right are the Consistency steps.

For short jobs it does not matter that much, but for alignments that take hours, 2 h vs 4 h makes a difference. And the longer the job, the longer the proportion of time spent waiting for the trailing threads seems to be. Is there a way to dynamically reassign parts of the work to cores that sit idle, or maybe before assigning the work, using some method to assess the "difficulty" of the alignment and weigh the work using this?

Thank you,
Lukasz

Oops. Workaround is to use muscle -h.

Hello,

Since only a Mac arm64 version is provided, I tried to build my own for a distribution on aarch64 ubuntu.

The build finished with zero errors, but when I ran muscle with no parameters I git this:

---Fatal error---
myutils.cpp(122) assert failed: sizeof(void *) == 4

hi @rcedgar
with muscle v5. How could i output aligned file with the clustalw or GCG MSC format.

Thanks a lot

Hi @rcedgar,
thanks for publishing MUSCLE on GitHub! I'm working on updating the MUSCLE package on Bioconda. Bioconda strongly recommends using releases (or at least Git tags) in package recipes, which is also general good practice for reproducibility. Would you please be able to tag a release on GitHub?

To align a pair of sequences of length L, muscle requires at least 5 x L^2 bytes of memory

The upper limit on L is currently a bit less than 30,000 letters. Unfortunately, this is close to the length of a Cov-2 genome, and an attempt to align Cov-2 genomes with muscle usually fails for this reason.

Currently, muscle segfaults if this limit is exceeded, this is a bug, it should give a graceful error message and exit.

This test command keeps prompting failure：
muscle -letterconf ensemble.efa -ref aln.afa -output letterconf.afa

Hello!

I have downloaded muscle version 5.1,linux_intel64 and ran alignment of this sequences file.

Command: ./muscle5.1.linux_intel64 -align corr_analysis/data/35_sequences.fasta -output corr_analysis/aligned_aligned_sequences.fa -refineiters 2
Output:

❯ ./muscle5.1.linux_intel64 -align corr_analysis/data/35_sequences.fasta -output corr_analysis/aligned_aligned_sequences.fa -refineiters 2

muscle 5.1.linux64 [12f0e2]  32.6Gb RAM, 12 cores
Built Jan 13 2022 23:17:13
(C) Copyright 2004-2021 Robert C. Edgar.
https://drive5.com

Input: 35 seqs, avg length 29834, max 31491

00:00 5.1Mb  CPU has 12 cores, running 12 threads
[1]    22676 segmentation fault (core dumped)  ./muscle5.1.linux_intel64 -align corr_analysis/data/35_sequences.fasta -outpu

Same issue happens with muscle_v5.0.1428_linux.

Is there any known workaround for the issue?

The version info for muscle5 is contained in ver_counter.h (referenced incorrectly in the code itself as ../ver/counter.h). This file contains a version string of 1219, while the executable in the binaries directory shows 1278. There are no tags or releases.

Please fix the file reference, preferably renaming it to the standard "version.h". Then tag and create a release.

The release is where the binaries should live, not in the repo. You will care if you are creating releases frequently and run out of push bandwidth.

There is still ample documentation on profile-profile alignment from v3.8, but searching the source code for v5 im not finding any references to it.. Has that been obsoleted or replaced by a new command?

Thanks a lot for your work.

Hello,

just a quick issue - the maxgapfract option doesn't seem to work:

muscle5 -resample sequences.ensemble.efa -maxgapfract 0.3 -output sequences.ensemble.gap03.efa

the output:

Invalid command line
Unknown option maxgapfract

Am I doing something wrong?

Thanks,
Lukasz

Error: Command-line option "ABC.fasta" must start with '-'

I am trying to align multiple fasta files together in one output file, but it is giving me the error above.

Hello! I downloaded the OSX binary but can not get it to run. My OSX version is macOS Monterey 12.0.1. Following the commands recommended in a closed issue, I get the following:

wget https://github.com/rcedgar/muscle/releases/download/v5.0.1428/muscle_v5.0.1428_osx
md5 muscle_v5.0.1428_osx
MD5 (muscle_v5.0.1428_osx) = d00d9f586476704f1a0f5a4fa1703b8c
./muscle_v5.0.1428_osx --version
dyld[88714]: missing symbol called
Abort trap: 6

wget https://github.com/rcedgar/muscle/releases/download/v5.0.1428/muscle_v5.0.1428_linux
chmod +x muscle_v5.0.1428_linux
$ muscle --h
FATAL: kernel too old
已放弃 (core dumped)

Dear Robert,

I am running muscle5 (binary or self-compiled binary) on a ubuntu 18.04 machine, and this error message appeared when trying to align more than 1 sequences

---Fatal error---
allocflat.cpp(15) assert failed: uint64(Size) == Size64---Fatal error---
allocflat.cpp(15) assert failed: uint64(Size) == Size64

The order is as follows,
muscle5 -super5 allde.fasta -output aln.afa

Best regards,
Runsheng

Hej Robert!
I'm running muscle5 on a computing cluster and all seems to work super smooth, but I've run into a small issue when trying to calculate the column confidence.

muscle -addconfseqs Gene_0107_diversified_ensemble.efa -output Gene_0107_diversified_ensemble_confseqs.efa

Below the output from the log file:

Invalid command line
Unknown option addconfseqs

muscle 5.1.linux64 [] 131Gb RAM, 20 cores
Built Mar 29 2022 15:27:13
(C) Copyright 2004-2021 Robert C. Edgar.
https://drive5.com

Is this an issue at my end or yours?
Cheers,
Karin

it happened after it was joining the
01:51:25 901Mb 100.0% Join 81 / 250 [1 x 717, 717 pairs]
01:51:54 901Mb 100.0% Join 82 / 250 [2 x 718, 1436 pairs]
01:52:05 901Mb 100.0% Join 83 / 250 [1 x 720, 720 pairs]
01:52:13 901Mb 100.0% Join 84 / 250 [1 x 721, 721 pairs]
01:52:14 901Mb 100.0% Join 85 / 250 [1 x 722, 722 pairs]
01:52:25 917Mb 100.0% Join 86 / 250 [2 x 723, 1446 pairs]
01:52:34 910Mb 100.0% Join 87 / 250 [2 x 725, 1450 pairs]
01:52:50 911Mb 100.0% Join 88 / 250 [5 x 727, 2000 pairs]
01:52:58 911Mb 100.0% Join 89 / 250 [1 x 732, 732 pairs]
01:53:10 910Mb 100.0% Join 90 / 250 [1 x 733, 733 pairs]
01:53:53 910Mb 100.0% Join 91 / 250 [5 x 734, 2000 pairs]
01:54:00 910Mb 100.0% Join 92 / 250 [1 x 739, 739 pairs]
01:54:05 910Mb 100.0% Join 93 / 250 [1 x 740, 740 pairs]
01:54:09 910Mb 100.0% Join 94 / 250 [1 x 741, 741 pairs]
01:54:14 910Mb 100.0% Join 95 / 250 [1 x 742, 742 pairs]
01:54:18 910Mb 100.0% Join 96 / 250 [1 x 743, 743 pairs]
01:54:46 910Mb 100.0% Join 97 / 250 [24 x 744, 2000 pairs]
01:54:53 911Mb 100.0% Join 98 / 250 [1 x 768, 768 pairs]
01:54:57 910Mb 100.0% Join 99 / 250 [1 x 769, 769 pairs]
01:55:05 910Mb 100.0% Join 100 / 250 [1 x 770, 770 pairs]
01:55:23 910Mb 100.0% Join 101 / 250 [1 x 771, 771 pairs]
01:55:39 912Mb 100.0% Join 102 / 250 [7 x 772, 2000 pairs]
01:56:11 910Mb 100.0% Join 103 / 250 [2 x 779, 1558 pairs]
01:56:26 910Mb 100.0% Join 104 / 250 [1 x 781, 781 pairs]
01:56:44 910Mb 100.0% Join 105 / 250 [2 x 782, 1564 pairs]
01:56:44 910Mb 100.0% Join 106 / 250 [1 x 2, 2 pairs]
01:56:44 910Mb 100.0% Join 107 / 250 [1 x 1, 1 pairs]
01:56:44 910Mb 100.0% Join 108 / 250 [1 x 2, 2 pairs]
01:56:44 929Mb 100.0% Join 109 / 250 [3 x 4, 12 pairs]
01:56:44 916Mb 100.0% Join 110 / 250 [2 x 7, 14 pairs]
01:56:44 916Mb 100.0% Join 111 / 250 [1 x 9, 9 pairs]
01:56:44 910Mb 100.0% Join 112 / 250 [1 x 2, 2 pairs]
01:56:57 910Mb 100.0% Join 113 / 250 [93 x 240, 2000 pairs]
01:57:11 910Mb 100.0% Join 114 / 250 [160 x 333, 2000 pairs]

muscle -super5 1_newfile.fasta -output aln.afa
Elapsed time 01:57:11
Max memory 1.2Gb

---Fatal error---
AssertSeqsEq E:\src\muscle5\pprog2.cpp:32

Dear developer,

From the manual https://drive5.com/muscle5/manual/cmd_super5.html, I noticed there is an option - Super5 - for aligning large sets of sequences. However, while testing with this option with the command muscleWin64.exe -super5 test.fasta -output alignedTest.fasta, it gave an error.

Error:

Invalid command line option "super5"

MUSCLE v3.8.31 by Robert C. Edgar

http://www.drive5.com/muscle
This software is donated to the public domain.
Please cite: Edgar, R.C. Nucleic Acids Res 32(5), 1792-97.


Basic usage

    muscle -in <inputfile> -out <outputfile>

Common options (for a complete list please see the User Guide):

    -in <inputfile>    Input file in FASTA format (default stdin)
    -out <outputfile>  Output alignment in FASTA format (default stdout)
    -diags             Find diagonals (faster for similar sequences)
    -maxiters <n>      Maximum number of iterations (integer, default 16)
    -maxhours <h>      Maximum time to iterate in hours (default no limit)
    -html              Write output in HTML format (default FASTA)
    -msf               Write output in GCG MSF format (default FASTA)
    -clw               Write output in CLUSTALW format (default FASTA)
    -clwstrict         As -clw, with 'CLUSTAL W (1.81)' header
    -log[a] <logfile>  Log to file (append if -loga, overwrite if -log)
    -quiet             Do not write progress messages to stderr
    -version           Display version information and exit

Without refinement (very fast, avg accuracy similar to T-Coffee): -maxiters 2
Fastest possible (amino acids): -maxiters 1 -diags -sv -distance1 kbit20_3
Fastest possible (nucleotides): -maxiters 1 -diags

I have tested run with the normal run with this command: `muscleWin64.exe -in test.fasta -out alignedTest.fasta. The tool is run properly. So, do you have any suggestions to solve this problem?

Best regards,
Li Chuin Chong

Make fails on recent gcc (>8, I think) unless this CFLAG is added

I have installed the most recent version of muscle with conda and from source and have run into the same issue.

I have a couple hundred thousand sequences so will potentially need to run with the -super5 argument. However, when I run with the -super5 algorithm, I get the following error:

muscle 5.1.linux64 [] 132Gb RAM, 24 cores

Input: 374249 seqs, length avg 1935 max 102417

WARNING: Sequence length >5k may require excessive memory
00:13 1.4Gb 100.0% Derep 335593 uniques, 38655 dupes
00:14 1.5Gb CPU has 24 cores, defaulting to 20 threads

muscle -super5 combined_seq.fasta -output super5_iter2.afa
Elapsed time 00:14

Max memory 1.5Gb
---Fatal error---
allocflat.cpp(15) assert failed: uint64(Size) == Size64

I should note that I am also running with -align which has not put out any issues.

Hi Robert,

Is it possible to keep the order of the sequences in the output file the same as in the input? (I've been getting this with the latest windows and linux pre-compiled binaries, with -align, with each producing a different, seemingly random order).

Cheers,
Seth

p.s. I've been really happy with the quality of alignments from muscle5!

Are you interested in a PR to set up Github CI for the build?

This is more of a question than an issue, but can you use muscle-align using stdin and stdout?

Thanks!