Error beta rarefaction plugin

Hi everyone! I have been trying to perform beta-rarefaction analyses without success. My efforts to try to debug the error have yielded little success so far. I am using qiime2-amplicon-2024.2 conda version. Please advice after going through the ddescription below.

Here is the code:

qiime diversity beta-rarefaction \
--i-table $QIIME_DENOISE_DIR/data_1_n_2/16S-feature_table_samoles.qza \
--p-metric weighted_unifrac \
--p-clustering-method nj \
--m-metadata-file $METADATA_4_RAREFACTION_DIR/data1_2_metadata.tsv \
--p-sampling-depth 225541 \
--i-phylogeny $PHYLOGENY_OUT_DIR/phylogenetic_tree/rooted_tree.qza \
--o-visualization $RAREFACTION_OUTPUT_DIR/beta_diversity/weighted_unifrac.qzv \
--verbose 

Here I use my --p-sampling-depth 225541 as suggested in previous forums about choosing sampling depths. I only have to samples to compare and I chose the lowest sampling depth.

Here is the log :

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/95c38cb8-7cc0-4570-bd7b-e2d6b31ddde6/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-30wo7yru

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/817637c7-b742-488c-b073-9450c94769eb/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-gmma1vuh

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/616c400a-6c48-4be7-a7c7-05a4407cfd18/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-2_aqjx8e

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/d99ee8a0-7083-4b8b-bbc7-73bb64162eb2/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-48nrz0n3

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/b07816b3-3f85-4bb2-b546-61990fe3eb1e/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-trspsfu0

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/2ebd55c9-c7a8-4a9d-a5bc-00f507f90b28/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-l9ow0a6j

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/67ea7d61-6de0-434d-aa81-a3dc5ad00214/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-ngj2rlwc

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a0434468-092e-46e2-a295-1065414e6f24/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-bsgwi2s1

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/eac2df15-a10f-4310-8a9a-c782f2d1f25a/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-5k5fq4sa

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/92810a7f-54c9-4d3f-9424-c152a3726a8d/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a912aff9-8f08-4016-b701-9cc6b4ada7b9/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-n_h4z4qf

Traceback (most recent call last):
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2cli/commands.py", line 520, in __call__
    results = self._execute_action(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2cli/commands.py", line 581, in _execute_action
    results = action(**arguments)
  File "<decorator-gen-246>", line 2, in beta_rarefaction
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
    outputs = self._callable_executor_(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 615, in _callable_executor_
    ret_val = self._callable(output_dir=temp_dir, **view_args)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity/_beta/_beta_rarefaction.py", line 67, in beta_rarefaction
    heatmap_fig, similarity_df = _make_heatmap(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity/_beta/_beta_rarefaction.py", line 115, in _make_heatmap
    sm_df = skbio.stats.distance.pwmantel(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/skbio/stats/distance/_mantel.py", line 605, in pwmantel
    stat, p_val, n = mantel(x, y, method=method, permutations=permutations,
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/skbio/stats/distance/_mantel.py", line 278, in mantel
    raise ValueError("Distance matrices must have at least 3 matching IDs "
ValueError: Distance matrices must have at least 3 matching IDs between them (i.e., minimum 3x3 in size).

Plugin error from diversity:

  Distance matrices must have at least 3 matching IDs between them (i.e., minimum 3x3 in size).

Running with the highest sampling depth gives a much weirder error:

qiime diversity beta-rarefaction --i-table $QIIME_DENOISE_DIR/data_1_n_2/16S-feature_table_samoles.qza --p-metric weighted_unifrac --p-clustering-method nj --m-metadata-file $METADATA_4_RAREFACTION_DIR/data1_2_metadata.tsv --p-sampling-depth 524879 --i-phylogeny $PHYLOGENY_OUT_DIR/phylogenetic_tree/rooted_tree.qza --o-visualization $RAREFACT
ION_OUTPUT_DIR/beta_diversity/weighted_unifrac.qzv --verbose
Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

ssu -i /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/3a4da24b-8c74-418c-a130-63d2b26266fa/data/feature-table.biom -t /var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a7098090-fd2b-42cf-a469-3dff8b2f921a/data/tree.nwk -m weighted_unnormalized -o /var/tmp/pbs.5614058.sched01/q2-LSMatFormat-wa80c8p5

*** Error in `/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2': free(): invalid next size (fast): 0x0000000001e6fa80 ***
======= Backtrace: =========
/lib64/libc.so.6(+0x7c503)[0x7ffff4d6e503]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x5e68df]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x683052]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x527918]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x518239]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x518f95]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x519389]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x4fd71b]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x56cd73]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x56f3ac]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x605d2d]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x539fa8]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x73202a]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x717e24]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x71ee78]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x53565b]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x5d5128]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x5d51e9]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x5d52bb]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x52d371]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x4e728a]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x4e7452]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x4398ce]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x4bf280]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x428459]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x42dc41]
/lib64/libc.so.6(__libc_start_main+0xf5)[0x7ffff4d13b35]
/home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2[0x4221f1]
======= Memory map: ========
00400000-011de000 r-xp 00000000 00:28 129206438                          /home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2
011de000-01213000 r-xp 00ddd000 00:28 129206438                          /home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2
01213000-0122c000 rwxp 00e12000 00:28 129206438                          /home/maloo/.conda/envs/qiime2-amplicon-2024.2/bin/ssu_nv_avx2
01400000-02665000 rwxp 00000000 00:00 0                                  [heap]
7ffff0000000-7ffff0021000 rwxp 00000000 00:00 0 
7ffff0021000-7ffff4000000 ---p 00000000 00:00 0 
7ffff4a14000-7ffff4c0b000 rwxp 00000000 00:00 0 
7ffff4c40000-7ffff4cd7000 rwxp 00000000 00:00 0 
7ffff4cd7000-7ffff4cf0000 r-xp 00000000 00:28 146980696                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libgcc_s.so.1
7ffff4cf0000-7ffff4cf1000 r-xp 00019000 00:28 146980696                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libgcc_s.so.1
7ffff4cf1000-7ffff4cf2000 rwxp 0001a000 00:28 146980696                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libgcc_s.so.1
7ffff4cf2000-7ffff4ea8000 r-xp 00000000 00:1c 151142                     /usr/lib64/libc-2.17.so
7ffff4ea8000-7ffff50a8000 ---p 001b6000 00:1c 151142                     /usr/lib64/libc-2.17.so
7ffff50a8000-7ffff50ac000 r-xp 001b6000 00:1c 151142                     /usr/lib64/libc-2.17.so
7ffff50ac000-7ffff50ae000 rwxp 001ba000 00:1c 151142                     /usr/lib64/libc-2.17.so
7ffff50ae000-7ffff50b3000 rwxp 00000000 00:00 0 
7ffff50b3000-7ffff5283000 r-xp 00000000 00:28 146972935                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libstdc++.so.6.0.32
7ffff5283000-7ffff528e000 r-xp 001cf000 00:28 146972935                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libstdc++.so.6.0.32
7ffff528e000-7ffff5292000 rwxp 001da000 00:28 146972935                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libstdc++.so.6.0.32
7ffff5292000-7ffff5297000 rwxp 00000000 00:00 0 
7ffff5297000-7ffff5397000 r-xp 00000000 00:1c 151581                     /usr/lib64/libm-2.17.so
7ffff5397000-7ffff5597000 ---p 00100000 00:1c 151581                     /usr/lib64/libm-2.17.so
7ffff5597000-7ffff5598000 r-xp 00100000 00:1c 151581                     /usr/lib64/libm-2.17.so
7ffff5598000-7ffff5599000 rwxp 00101000 00:1c 151581                     /usr/lib64/libm-2.17.so
7ffff5599000-7ffff559b000 r-xp 00000000 00:1c 151201                     /usr/lib64/libdl-2.17.so
7ffff559b000-7ffff579b000 ---p 00002000 00:1c 151201                     /usr/lib64/libdl-2.17.so
7ffff579b000-7ffff579c000 r-xp 00002000 00:1c 151201                     /usr/lib64/libdl-2.17.so
7ffff579c000-7ffff579d000 rwxp 00003000 00:1c 151201                     /usr/lib64/libdl-2.17.so
7ffff579d000-7ffff57b6000 r-xp 00000000 00:28 146992527                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libz.so.1.2.13
7ffff57b6000-7ffff57b7000 r-xp 00018000 00:28 146992527                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libz.so.1.2.13
7ffff57b7000-7ffff57b8000 rwxp 00019000 00:28 146992527                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libz.so.1.2.13
7ffff57b8000-7ffff57bf000 r-xp 00000000 00:1c 151737                     /usr/lib64/librt-2.17.so
7ffff57bf000-7ffff59be000 ---p 00007000 00:1c 151737                     /usr/lib64/librt-2.17.so
7ffff59be000-7ffff59bf000 r-xp 00006000 00:1c 151737                     /usr/lib64/librt-2.17.so
7ffff59bf000-7ffff59c0000 rwxp 00007000 00:1c 151737                     /usr/lib64/librt-2.17.so
7ffff59c0000-7ffff59d7000 r-xp 00000000 00:1c 151709                     /usr/lib64/libpthread-2.17.so
7ffff59d7000-7ffff5bd6000 ---p 00017000 00:1c 151709                     /usr/lib64/libpthread-2.17.so
7ffff5bd6000-7ffff5bd7000 r-xp 00016000 00:1c 151709                     /usr/lib64/libpthread-2.17.so
7ffff5bd7000-7ffff5bd8000 rwxp 00017000 00:1c 151709                     /usr/lib64/libpthread-2.17.so
7ffff5bd8000-7ffff5bdc000 rwxp 00000000 00:00 0 
7ffff5bdc000-7ffff5c13000 r-xp 00000000 00:28 146980702                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libquadmath.so.0.0.0
7ffff5c13000-7ffff5c14000 r-xp 00036000 00:28 146980702                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libquadmath.so.0.0.0
7ffff5c14000-7ffff5c15000 rwxp 00037000 00:28 146980702                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libquadmath.so.0.0.0
7ffff5c15000-7ffff5d36000 r-xp 00000000 00:28 147002066                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libopenblasp-r0.3.25.so
7ffff5d36000-7ffff5d3d000 ---p 00121000 00:28 147002066                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libopenblasp-r0.3.25.so
7ffff5d3d000-7ffff7dac000 r-xp 00128000 00:28 147002066                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libopenblasp-r0.3.25.so
7ffff7dac000-7ffff7dba000 r-xp 02197000 00:28 147002066                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libopenblasp-r0.3.25.so
7ffff7dba000-7ffff7dcc000 rwxp 021a5000 00:28 147002066                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libopenblasp-r0.3.25.so
7ffff7dcc000-7ffff7ddd000 rwxp 00000000 00:00 0 
7ffff7ddd000-7ffff7dfd000 r-xp 00000000 00:1c 150999                     /usr/lib64/ld-2.17.so
7ffff7dfd000-7ffff7dfe000 rwxp 00000000 00:00 0 
7ffff7dfe000-7ffff7e09000 r-xp 00000000 00:28 94888172                   /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libsz.so.2.0.1
7ffff7e09000-7ffff7e0a000 r-xp 0000a000 00:28 94888172                   /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libsz.so.2.0.1
7ffff7e0a000-7ffff7e0b000 rwxp 0000b000 00:28 94888172                   /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libsz.so.2.0.1
7ffff7e0b000-7ffff7e0c000 rwxp 00000000 00:00 0 
7ffff7e21000-7ffff7e22000 rwxp 00000000 00:00 0 
7ffff7e22000-7ffff7fca000 r-xp 00000000 00:28 146991959                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libgfortran.so.5.0.0
7ffff7fca000-7ffff7fcb000 r-xp 001a8000 00:28 146991959                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libgfortran.so.5.0.0
7ffff7fcb000-7ffff7fcd000 rwxp 001a9000 00:28 146991959                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/libgfortran.so.5.0.0
7ffff7fcd000-7ffff7fce000 rwxp 00000000 00:00 0 
7ffff7fce000-7ffff7ff7000 r-xp 00000000 00:28 146992538                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/liblz4.so.1.9.4
7ffff7ff7000-7ffff7ff8000 r-xp 00028000 00:28 146992538                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/liblz4.so.1.9.4
7ffff7ff8000-7ffff7ff9000 rwxp 00029000 00:28 146992538                  /home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/liblz4.so.1.9.4
7ffff7ff9000-7ffff7ffa000 rwxp 00000000 00:00 0 
7ffff7ffa000-7ffff7ffc000 r-xp 00000000 00:00 0                          [vdso]
7ffff7ffc000-7ffff7ffd000 r-xp 0001f000 00:1c 150999                     /usr/lib64/ld-2.17.so
7ffff7ffd000-7ffff7ffe000 rwxp 00020000 00:1c 150999                     /usr/lib64/ld-2.17.so
7ffff7ffe000-7ffff7fff000 rwxp 00000000 00:00 0 
7ffffffdb000-7ffffffff000 rwxp 00000000 00:00 0                          [stack]
ffffffffff600000-ffffffffff601000 r-xp 00000000 00:00 0                  [vsyscall]
Traceback (most recent call last):
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2cli/commands.py", line 520, in __call__
    results = self._execute_action(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2cli/commands.py", line 581, in _execute_action
    results = action(**arguments)
  File "<decorator-gen-246>", line 2, in beta_rarefaction
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
    outputs = self._callable_executor_(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 615, in _callable_executor_
    ret_val = self._callable(output_dir=temp_dir, **view_args)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity/_beta/_beta_rarefaction.py", line 61, in beta_rarefaction
    distance_matrices = _get_multiple_rarefaction(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity/_beta/_beta_rarefaction.py", line 107, in _get_multiple_rarefaction
    distance_matrix, = beta_func(table=rarefied_table, metric=metric)
  File "<decorator-gen-819>", line 2, in beta_phylogenetic
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/context.py", line 143, in deferred_action
    return action_obj._bind(
  File "<decorator-gen-823>", line 2, in beta_phylogenetic
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
    outputs = self._callable_executor_(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 647, in _callable_executor_
    outputs = self._callable(scope.ctx, **view_args)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity/_beta/_pipeline.py", line 31, in beta_phylogenetic
    dm, = action(table, phylogeny, threads=threads,
  File "<decorator-gen-824>", line 2, in weighted_unifrac
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/context.py", line 143, in deferred_action
    return action_obj._bind(
  File "<decorator-gen-825>", line 2, in weighted_unifrac
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
    outputs = self._callable_executor_(
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 566, in _callable_executor_
    output_views = self._callable(**view_args)
  File "<decorator-gen-145>", line 2, in weighted_unifrac
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 75, in _validate_tables
    return wrapped_function(*args, **kwargs)
  File "<decorator-gen-144>", line 2, in weighted_unifrac
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 118, in _validate_requested_cpus
    return wrapped_function(*bound_arguments.args, **bound_arguments.kwargs)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity_lib/beta.py", line 243, in weighted_unifrac
    _omp_cmd_wrapper(threads, cmd)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 134, in _omp_cmd_wrapper
    return _run_external_cmd(cmd, verbose=verbose, env=env)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 128, in _run_external_cmd
    return subprocess.run(cmd, check=True, env=env)
  File "/home/maloo/.conda/envs/qiime2-amplicon-2024.2/lib/python3.8/subprocess.py", line 516, in run
    raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['ssu', '-i', '/var/tmp/pbs.5614058.sched01/qiime2/maloo/data/3a4da24b-8c74-418c-a130-63d2b26266fa/data/feature-table.biom', '-t', '/var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a7098090-fd2b-42cf-a469-3dff8b2f921a/data/tree.nwk', '-m', 'weighted_unnormalized', '-o', '/var/tmp/pbs.5614058.sched01/q2-LSMatFormat-wa80c8p5']' died with <Signals.SIGABRT: 6>.

Plugin error from diversity:

  Command '['ssu', '-i', '/var/tmp/pbs.5614058.sched01/qiime2/maloo/data/3a4da24b-8c74-418c-a130-63d2b26266fa/data/feature-table.biom', '-t', '/var/tmp/pbs.5614058.sched01/qiime2/maloo/data/a7098090-fd2b-42cf-a469-3dff8b2f921a/data/tree.nwk', '-m', 'weighted_unnormalized', '-o', '/var/tmp/pbs.5614058.sched01/q2-LSMatFormat-wa80c8p5']' died with <Signals.SIGABRT: 6>.

See above for debug info.

Here is my featuretable qzv:
16S-feature_table_samoles2.qzv (586.6 KB)

Here is my metadata

sample-id	source	subCounty	studySIte	fertiliserUsage
S1-S1-L001-001	soil	Gatundu-North	Gituamba	Yes
S2-S1-L001-001 	soil	Gatundu-North	Mangu	        No

Hi @George_kitundu,
According to the first error:

Distance matrices must have at least 3 matching IDs between them (i.e., minimum 3x3 in size).

Looking at your feature table, you only have 2 samples. So it looks like you won't be a ble to run beta rarefaction because your data does not meet the minimum required samples for beta-rarefaction.

I am sorry to not have better news! I hope this clarifies your error!

:turtle:

1 Like

Thanks a lot, @cherman2 , I am in a bit of peril here haha. Unfortunately, my two collaborators had a bit of a misunderstanding and one sequenced the two 16S and the other sequenced the remaining sample shotgun. I conducted the analysis using qiime2. I am a bit skeptical about using the shotgun version since it says somewhere that its still in its testing stage. How would you advise me to go about this issue so I can perform the rarefaction analyses in all three samples? Could I just use the qiime2 shotgun feature and find a way to combine the feature tables and phylogenies for all the three samples then calculate the rarefaction analyses?

Hello George,

I'm not Cherman2 but hopefully I can offer a little advice.

It sounds like you have a total of 3 samples now. How many samples will you have in total?

If this is a pilot study, having both shotgun and amplicon samples can be a good starting point. However, you could also use data from another study. If you want to run the Qiime2 pipeline to learn how analysis works and test that your system has everything installed, you can use the data from one of the tutorials.

I understand the struggle of getting data from collaborators, and how our colleagues can make mistakes. Hopefully, we can make a plan that works in the future!

I would not. Amplicon and shotgun sequencing are quite different and I suspect reviewer three will criticize this.

1 Like

Thanks a lot for the input @colinbrislawn , I will have a total of three samples . All of which come from different locations. Two of them are the 16S an one is shotgun.

I am really perplexed on what to do because whats a metagenome study without the rarefactions haha

Okay. So you have a global n = 3, and n=1 for each location.

This may work as a demonstration or proof-of-concept, but there is insufficient statistical power to draw any conclusions.

Your team needs more samples.

Don't feel too bad: NASA has done studies with similarly small sample sizes. They argue that this is due to the rarity of their cohort

long-duration (>300 days) missions are rare (n = 8 total)

Even this study is longitudinal, so even they use more than 3 samples.


The rarefaction curves show when sufficient sampling depth (reads per sample) has been reached.

This study may have enough reads per sample, but it does not have enough samples.

Your team needs more samples.

3 Likes

Well thats a relief, I managed to plot the alpha rarefactions just fine, core metrics came out well as well. My only concern is with the beta rarefaction which is in my opinion, kinda the whole idea when studying different sites/locations

1 Like