msa Error

[rwilton1]

Hello,

I'm failing to complete the msa step in trycycler. I've copied the message below. I'm wondering if the issue might be with large number of repeats (~20; ~70Kb total length) in my engineered genome. To provide some wiggle room for repeat issues, I allowed the max_indel size to be 2000 (Indel sizes are listed at bottom). Thanks for any advice!

Error message:*********************************************************************
Merging MSA (2022-11-08 18:52:31)
Each of the MSA pieces are now merged together and saved to file.

Traceback (most recent call last):
File "/homes/rwilton/software/miniconda3/envs/nano-trycycler/bin/trycycler", line 10, in
sys.exit(main())
File "/homes/rwilton/software/miniconda3/envs/nano-trycycler/lib/python3.10/site-packages/trycycler/main.py", line 51, in main
msa(args)
File "/homes/rwilton/software/miniconda3/envs/nano-trycycler/lib/python3.10/site-packages/trycycler/msa.py", line 36, in msa
merge_pieces(temp_dir, args.cluster_dir, seqs)
File "/homes/rwilton/software/miniconda3/envs/nano-trycycler/lib/python3.10/site-packages/trycycler/msa.py", line 175, in merge_pieces
aligned_seq_parts[n].append(parts[n].upper())
KeyError: 'A_contig_1'

Indels from reconcile step**********************************************************
A_contig_1 vs B_contig_1... 99.00% identity, max indel = 43
A_contig_1 vs C_contig_1... 99.93% identity, max indel = 27
A_contig_1 vs D_contig_1... 99.78% identity, max indel = 37
A_contig_1 vs E_Utg648... 99.88% identity, max indel = 604
A_contig_1 vs G_Utg580... 99.72% identity, max indel = 301
A_contig_1 vs H_Utg592... 99.93% identity, max indel = 781
A_contig_1 vs L_utg000001l... 99.18% identity, max indel = 39
B_contig_1 vs C_contig_1... 99.07% identity, max indel = 1452
B_contig_1 vs D_contig_1... 99.21% identity, max indel = 1669
B_contig_1 vs E_Utg648... 98.89% identity, max indel = 1533
B_contig_1 vs G_Utg580... 99.17% identity, max indel = 1521
B_contig_1 vs H_Utg592... 98.97% identity, max indel = 1704
B_contig_1 vs L_utg000001l... 99.77% identity, max indel = 94
C_contig_1 vs D_contig_1... 99.86% identity, max indel = 37
C_contig_1 vs E_Utg648... 99.81% identity, max indel = 1401
C_contig_1 vs G_Utg580... 99.79% identity, max indel = 301
C_contig_1 vs H_Utg592... 99.88% identity, max indel = 781
C_contig_1 vs L_utg000001l... 99.25% identity, max indel = 40
D_contig_1 vs E_Utg648... 99.67% identity, max indel = 1156
D_contig_1 vs G_Utg580... 99.93% identity, max indel = 301
D_contig_1 vs H_Utg592... 99.75% identity, max indel = 1499
D_contig_1 vs L_utg000001l... 99.39% identity, max indel = 38
E_Utg648 vs G_Utg580... 99.64% identity, max indel = 301
E_Utg648 vs H_Utg592... 99.84% identity, max indel = 781
E_Utg648 vs L_utg000001l... 99.09% identity, max indel = 274
G_Utg580 vs H_Utg592... 99.73% identity, max indel = 1499
G_Utg580 vs L_utg000001l... 99.38% identity, max indel = 301
H_Utg592 vs L_utg000001l... 99.18% identity, max indel = 599

[Aluminio-visto]

Hi, I'm having a similar issue in this step, but in our case we have a wild-type Pseudomonas genome:
Merging MSA (2022-12-07 11:33:28)
Each of the MSA pieces are now merged together and saved to file.

Traceback (most recent call last):
File "/home/usuario/miniconda3/envs/trycycler/bin/trycycler", line 10, in
sys.exit(main())
File "/home/usuario/miniconda3/envs/trycycler/lib/python3.10/site-packages/trycycler/main.py", line 51, in main
msa(args)
File "/home/usuario/miniconda3/envs/trycycler/lib/python3.10/site-packages/trycycler/msa.py", line 36, in msa
merge_pieces(temp_dir, args.cluster_dir, seqs)
File "/home/usuario/miniconda3/envs/trycycler/lib/python3.10/site-packages/trycycler/msa.py", line 175, in merge_pieces
aligned_seq_parts[n].append(parts[n].upper())
KeyError: 'A_contig_2'

Any help will be very much appreciated

[uyghbo]

Dear Trycycler Team

Got the same error here for a 5.4 MB Klebsiella pneumoniae genome

ESC[93mESC[1mESC[4mMerging MSAESC[0m ESC[2m(2022-12-30 15:38:36)ESC[0m
ESC[2m Each of the MSA pieces are now merged together and saved to file.ESC[0m

Traceback (most recent call last):
File "/home/miniconda3/envs/bactopia/envs/unicycler/bin/trycycler", line 10, in
sys.exit(main())
File "/home/miniconda3/envs/unicycler/lib/python3.10/site-packages/trycycler/main.py", line 51, in main
msa(args)
File "/home/miniconda3/envs/unicycler/lib/python3.10/site-packages/trycycler/msa.py", line 36, in msa
merge_pieces(temp_dir, args.cluster_dir, seqs)
File "/home/miniconda3/envs/unicycler/lib/python3.10/site-packages/trycycler/msa.py", line 175, in merge_pieces
aligned_seq_parts[n].append(parts[n].upper())
KeyError: 'E_ctg000030'

[TheIncredibleMulk]

Dear Trycycler Team,

Got a similar error for a 5MB Acidobacteriota Genome

Traceback (most recent call last):
  File "/home/seq/miniconda3/envs/trycycler/bin/trycycler", line 10, in <module>
    sys.exit(main())
  File "/home/seq/miniconda3/envs/trycycler/lib/python3.10/site-packages/trycycler/main.py", line 51, in main
    msa(args)
  File "/home/seq/miniconda3/envs/trycycler/lib/python3.10/site-packages/trycycler/msa.py", line 36, in msa
    merge_pieces(temp_dir, args.cluster_dir, seqs)
  File "/home/seq/miniconda3/envs/trycycler/lib/python3.10/site-packages/trycycler/msa.py", line 175, in merge_pieces
    aligned_seq_parts[n].append(parts[n].upper())
KeyError: 'A_contig_1'

Thanks in advance for any help or guidance correcting the issue.

[TheIncredibleMulk]

Updating my previous issue. Backdating muscle to version 3.8.1551 fixed the issue and it ran fine. Hopefully that helps others with this issue.

[rmormando]

how did you backdate it?

[TheIncredibleMulk]

Depends on how you installed it. If you used conda like I did then run the following.
Activate the trycycler environment.
conda activate trycycler
Backdate your version of muscle.
conda install muscle=3.8.1551

An additional note, depending on the flavor of Linux / environment you're running I've had to use different versions but 3.8.1551 seemed the most consistent, but I also had success in some systems with 3.8.31 .

[rmormando]

That worked!! Thank you!!

[rrwick]

Thanks, all! I've updated Trycycler's docs to make it clear that MUSCLE v3 is strongly recommended. And after the next release of Trycycler (soon), I'll ensure that the conda recipe has muscle<4.

Ryan