Trouble with assembly of unusually long chloroplast genomes

[Tmesipteris]

I am having trouble assembling the large (around 200,000 bp) chloroplast genome of a Rhododendron species. I cannot get the complete circular output using the recommended settings that work well for other species I am investigating with normal size (around 150,000 bp) cp genomes. Could I please have some advice on adjusting the settings to improve the result?

Here is the log of the failed run for Rhododendron:

get_organelle_from_reads.py -1 Rbr53_560_1.fq.gz -2 Rbr53_560_2.fq.gz -s MN413198.fasta -o Rbr53_output -R 15 -k 21,45,65,85,105 -F embplant_pt

2022-06-15 14:30:24,647 - INFO: Pre-reading fastq ...
2022-06-15 14:30:24,648 - INFO: Estimating reads to use ... (to use all reads, set '--reduce-reads-for-coverage inf')
2022-06-15 14:30:24,897 - INFO: Tasting 100000+100000 reads ...
2022-06-15 14:31:22,467 - INFO: Estimating reads to use finished.
2022-06-15 14:31:22,469 - INFO: Unzipping reads file: Rbr53_560_1.fq.gz (537525581 bytes)
2022-06-15 14:37:40,360 - INFO: Unzipping reads file: Rbr53_560_2.fq.gz (564282312 bytes)
2022-06-15 14:44:38,576 - INFO: Counting read qualities ...
2022-06-15 14:44:38,727 - INFO: Identified quality encoding format = Sanger
2022-06-15 14:44:38,728 - INFO: Trimming bases with qualities (0.00%): 33..33 !
2022-06-15 14:44:38,806 - INFO: Mean error rate = 0.0036
2022-06-15 14:44:38,808 - INFO: Counting read lengths ...
2022-06-15 14:44:50,414 - INFO: Mean = 150.0 bp, maximum = 150 bp.
2022-06-15 14:44:50,414 - INFO: Reads used = 4004045+4004045
2022-06-15 14:44:50,415 - INFO: Pre-reading fastq finished.

2022-06-15 14:44:50,416 - INFO: Making seed reads ...
2022-06-15 14:44:50,821 - INFO: Making seed - bowtie2 index ...
2022-06-15 14:44:56,963 - INFO: Making seed - bowtie2 index finished.
2022-06-15 14:44:56,964 - INFO: Mapping reads to seed bowtie2 index ...
2022-06-15 14:56:57,858 - INFO: Mapping finished.
2022-06-15 14:56:57,860 - INFO: Seed reads made: Rbr53_using_whole_cp_as_ref_output/seed/embplant_pt.initial.fq (119225830 bytes)
2022-06-15 14:56:57,862 - INFO: Making seed reads finished.

2022-06-15 14:56:57,862 - INFO: Checking seed reads and parameters ...
2022-06-15 14:56:57,863 - INFO: The automatically-estimated parameter(s) do not ensure the best choice(s).
2022-06-15 14:56:57,863 - INFO: If the result graph is not a circular organelle genome,
2022-06-15 14:56:57,864 - INFO: you could adjust the value(s) of '-w'/'-R' for another new run.
2022-06-15 14:57:15,665 - INFO: Pre-assembling mapped reads ...
2022-06-15 14:58:31,580 - INFO: Pre-assembling mapped reads finished.
2022-06-15 14:58:31,581 - INFO: Estimated embplant_pt-hitting base-coverage = 282.56
2022-06-15 14:58:32,191 - INFO: Estimated word size(s): 112
2022-06-15 14:58:32,192 - INFO: Setting '-w 112'
2022-06-15 14:58:32,193 - INFO: Setting '--max-extending-len inf'
2022-06-15 14:58:32,933 - INFO: Checking seed reads and parameters finished.

2022-06-15 14:58:32,933 - INFO: Making read index ...
2022-06-15 15:06:26,936 - INFO: 7573209 candidates in all 8008090 reads
2022-06-15 15:06:26,955 - INFO: Pre-grouping reads ...
2022-06-15 15:06:26,955 - INFO: Setting '--pre-w 112'
2022-06-15 15:06:27,655 - INFO: 200000/270637 used/duplicated
2022-06-15 15:06:43,939 - INFO: 7292 groups made.
2022-06-15 15:06:45,327 - INFO: Making read index finished.

2022-06-15 15:06:45,328 - INFO: Extending ...
2022-06-15 15:06:45,329 - INFO: Adding initial words ...
2022-06-15 15:06:55,072 - INFO: AW 5080954
2022-06-15 15:07:35,547 - INFO: Round 1: 7573209/7573209 AI 205558 AW 5119068
2022-06-15 15:08:11,991 - INFO: Round 2: 7573209/7573209 AI 206157 AW 5127438
2022-06-15 15:08:48,411 - INFO: Round 3: 7573209/7573209 AI 206693 AW 5133288
2022-06-15 15:09:24,838 - INFO: Round 4: 7573209/7573209 AI 207227 AW 5139558
2022-06-15 15:10:01,614 - INFO: Round 5: 7573209/7573209 AI 207898 AW 5146738
2022-06-15 15:10:38,147 - INFO: Round 6: 7573209/7573209 AI 208499 AW 5152344
2022-06-15 15:11:14,924 - INFO: Round 7: 7573209/7573209 AI 209029 AW 5157826
2022-06-15 15:11:51,362 - INFO: Round 8: 7573209/7573209 AI 209535 AW 5163286
2022-06-15 15:12:27,785 - INFO: Round 9: 7573209/7573209 AI 210103 AW 5170144
2022-06-15 15:13:04,193 - INFO: Round 10: 7573209/7573209 AI 210769 AW 5176484
2022-06-15 15:13:40,604 - INFO: Round 11: 7573209/7573209 AI 211342 AW 5181980
2022-06-15 15:14:17,052 - INFO: Round 12: 7573209/7573209 AI 211879 AW 5188364
2022-06-15 15:14:53,495 - INFO: Round 13: 7573209/7573209 AI 212475 AW 5195398
2022-06-15 15:15:29,948 - INFO: Round 14: 7573209/7573209 AI 213107 AW 5201738
2022-06-15 15:16:06,384 - INFO: Round 15: 7573209/7573209 AI 213656 AW 5208184
2022-06-15 15:16:06,384 - INFO: Hit the round limit 15 and terminated ...
2022-06-15 15:16:34,209 - INFO: Extending finished.

2022-06-15 15:16:34,214 - INFO: Separating filtered fastq file ...
2022-06-15 15:22:03,178 - INFO: Setting '-k 21,45,65,85,105'
2022-06-15 15:22:03,179 - INFO: Assembling using SPAdes ...
2022-06-15 15:22:03,282 - INFO: spades.py -t 1 -1 Rbr53_using_whole_cp_as_ref_output/filtered_1_paired.fq -2 Rbr53_using_whole_cp_as_ref_output/filtered_2_paired.fq --s1 Rbr53_using_whole_cp_as_ref_output/filtered_1_unpaired.fq --s2 Rbr53_using_whole_cp_as_ref_output/filtered_2_unpaired.fq -k 21,45,65,85,105 -o Rbr53_using_whole_cp_as_ref_output/filtered_spades
2022-06-15 15:27:39,777 - INFO: Insert size = 272.814, deviation = 57.6943, left quantile = 200, right quantile = 348
2022-06-15 15:27:39,778 - INFO: Assembling finished.

2022-06-15 15:27:41,976 - INFO: Slimming Rbr53_using_whole_cp_as_ref_output/filtered_spades/K105/assembly_graph.fastg finished!
2022-06-15 15:27:43,738 - INFO: Slimming Rbr53_using_whole_cp_as_ref_output/filtered_spades/K85/assembly_graph.fastg finished!
2022-06-15 15:27:45,506 - INFO: Slimming Rbr53_using_whole_cp_as_ref_output/filtered_spades/K65/assembly_graph.fastg finished!
2022-06-15 15:27:47,280 - INFO: Slimming Rbr53_using_whole_cp_as_ref_output/filtered_spades/K45/assembly_graph.fastg finished!
2022-06-15 15:27:47,281 - INFO: Slimming assembly graphs finished.

2022-06-15 15:27:47,285 - INFO: Extracting embplant_pt from the assemblies ...
2022-06-15 15:27:47,293 - INFO: Disentangling Rbr53_using_whole_cp_as_ref_output/filtered_spades/K105/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2022-06-15 15:27:50,926 - INFO: Disentangling failed: 'Incomplete/Complicated graph: please check around EDGE_102796!'
2022-06-15 15:27:50,928 - INFO: Disentangling Rbr53_using_whole_cp_as_ref_output/filtered_spades/K85/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2022-06-15 15:27:52,180 - INFO: Disentangling failed: 'Multiple isolated embplant_pt components detected! Broken or contamination?'
2022-06-15 15:27:52,182 - INFO: Disentangling Rbr53_using_whole_cp_as_ref_output/filtered_spades/K65/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2022-06-15 15:27:54,274 - WARNING: The graph might suffer from contamination or polymorphism!
2022-06-15 15:27:54,275 - WARNING: Only the contig with the max cov was kept for each of those 1 polymorphic loci.
2022-06-15 15:28:10,662 - INFO: Disentangling failed: 'Free variable > 10 is not accepted yet!'
2022-06-15 15:28:10,664 - INFO: Disentangling Rbr53_using_whole_cp_as_ref_output/filtered_spades/K45/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2022-06-15 15:28:12,173 - INFO: Disentangling failed: 'Incomplete/Complicated graph: please check around EDGE_29282!'
2022-06-15 15:28:12,175 - INFO: Scaffolding disconnected contigs using SPAdes scaffolds ...
2022-06-15 15:28:12,176 - WARNING: Assembly based on scaffolding may not be as accurate as the ones directly exported from the assembly graph.
2022-06-15 15:28:12,177 - INFO: Disentangling Rbr53_using_whole_cp_as_ref_output/filtered_spades/K105/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2022-06-15 15:28:12,191 - WARNING: Connection between 99424_head and 99116_tail already existed!
2022-06-15 15:28:12,192 - WARNING: Connection between 17430_tail and 98056_head already existed!
2022-06-15 15:28:12,192 - INFO: Disentangling failed: 'No new connections.'
2022-06-15 15:28:12,194 - INFO: Disentangling Rbr53_using_whole_cp_as_ref_output/filtered_spades/K105/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a/an embplant_pt-insufficient graph ...
2022-06-15 15:28:15,284 - INFO: Average embplant_pt kmer-coverage = 99.4
2022-06-15 15:28:15,285 - INFO: Average embplant_pt base-coverage = 324.2
2022-06-15 15:28:15,285 - INFO: Writing output ...
2022-06-15 15:28:15,341 - INFO: Writing PATH1 of embplant_pt scaffold(s) to Rbr53_using_whole_cp_as_ref_output/embplant_pt.K105.scaffolds.graph1.1.path_sequence.fasta
2022-06-15 15:28:15,348 - INFO: Writing GRAPH to Rbr53_using_whole_cp_as_ref_output/embplant_pt.K105.contigs.graph1.selected_graph.gfa
2022-06-15 15:28:15,435 - INFO: Result status of embplant_pt: 14 scaffold(s)
2022-06-15 15:28:15,726 - INFO: Writing output finished.
2022-06-15 15:28:15,729 - INFO: Please ...
2022-06-15 15:28:15,730 - INFO: load the graph file 'assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg' in K105,K85,K65,K45
2022-06-15 15:28:15,731 - INFO: load the CSV file 'assembly_graph.fastg.extend-embplant_pt-embplant_mt.csv' in K105,K85,K65,K45
2022-06-15 15:28:15,731 - INFO: visualize and confirm the incomplete result in Bandage.
2022-06-15 15:28:15,732 - INFO: If the result is nearly complete,
2022-06-15 15:28:15,733 - INFO: you can also adjust the arguments to try again.
2022-06-15 15:28:15,733 - INFO: If you have questions for us, please provide us with the get_org.log.txt file and the post-slimming graph in the format you like!
2022-06-15 15:28:15,734 - INFO: Extracting embplant_pt from the assemblies finished.

Total cost 3473.60 s
Thank you!

[Hua-CM]

I seems encounter the same problem, any advices?

[Kinggerm]

@Hua-CM Please see a duplicated thread #256 for the reply and leave your further comments there.
This thread will be transferred and locked.