msa Error

[rowi2024]

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