A question about running getorganlle

[Mingyu0110]

I used getorganelle to assemble the chloroplast genome. Run the command as follows:

get_organelle_from_reads.py -1 ../data/V1_1.fq.gz -2 ../data/V1_2.fq.gz -o V1 -R 30 -k 21,45,65,85,105,127 -t 20 -F embplant_pt

Some samples were not obtained with complete chloroplast assembly.

Can you please tell me how to adjust the parameters to get complete chloroplast assembly？The log file is as follows：

GetOrganelle v1.7.7.0

get_organelle_from_reads.py assembles organelle genomes from genome skimming data.
Find updates in https://github.com/Kinggerm/GetOrganelle and see README.md for more information.

Python 3.9.9 | packaged by conda-forge | (main, Dec 20 2021, 02:41:03) [GCC 9.4.0]
PLATFORM: Linux lzu-MZ72-HB0-00 5.4.0-132-generic #148~18.04.1-Ubuntu SMP Mon Oct 24 20:41:14 UTC 2022 x86_64 x86_64
PYTHON LIBS: GetOrganelleLib 1.7.7.0; numpy 1.22.3; sympy 1.11.1; scipy 1.9.3
DEPENDENCIES: Bowtie2 2.4.5; SPAdes 3.15.5; Blast 2.11.0
GETORG_PATH=/home/limy/.GetOrganelle
SEED DB: embplant_pt 0.0.1; embplant_mt 0.0.1
LABEL DB: embplant_pt 0.0.1; embplant_mt 0.0.1
WORKING DIR: /mnt/sdb/limy/workspace/1.variant_calling/chloroplast3
//mnt/sdb/limy/bin/miniconda3/bin/get_organelle_from_reads.py -1 ../data/V1_1.fq.gz -2 ../data/V1_2.fq.gz -o V1 -R 30 -k 21,45,65,85,105,127 -t 20 -F embplant_pt

2023-02-14 14:27:03,272 - INFO: Pre-reading fastq ...
2023-02-14 14:27:03,272 - INFO: Estimating reads to use ... (to use all reads, set '--reduce-reads-for-coverage inf --max-reads inf')
2023-02-14 14:27:03,345 - INFO: Tasting 100000+100000 reads ...
2023-02-14 14:27:13,691 - INFO: Tasting 500000+500000 reads ...
2023-02-14 14:27:30,695 - INFO: Estimating reads to use finished.
2023-02-14 14:27:30,695 - INFO: Unzipping reads file: ../data/V1_1.fq.gz (4843326106 bytes)
2023-02-14 14:27:59,958 - INFO: Unzipping reads file: ../data/V1_2.fq.gz (5175943520 bytes)
2023-02-14 14:28:31,297 - INFO: Counting read qualities ...
2023-02-14 14:28:31,447 - INFO: Identified quality encoding format = Sanger
2023-02-14 14:28:31,448 - INFO: Phred offset = 33
2023-02-14 14:28:31,450 - INFO: Trimming bases with qualities (0.00%): 33..33 !
2023-02-14 14:28:31,499 - INFO: Mean error rate = 0.0026
2023-02-14 14:28:31,501 - INFO: Counting read lengths ...
2023-02-14 14:29:16,774 - INFO: Mean = 150.0 bp, maximum = 150 bp.
2023-02-14 14:29:16,774 - INFO: Reads used = 15000000+15000000
2023-02-14 14:29:16,774 - INFO: Pre-reading fastq finished.

2023-02-14 14:29:16,774 - INFO: Making seed reads ...
2023-02-14 14:29:16,775 - INFO: Seed bowtie2 index existed!
2023-02-14 14:29:16,775 - INFO: Mapping reads to seed bowtie2 index ...
2023-02-14 14:34:57,359 - INFO: Mapping finished.
2023-02-14 14:34:57,361 - INFO: Seed reads made: V1/seed/embplant_pt.initial.fq (65705782 bytes)
2023-02-14 14:34:57,361 - INFO: Making seed reads finished.

2023-02-14 14:34:57,361 - INFO: Checking seed reads and parameters ...
2023-02-14 14:34:57,361 - INFO: The automatically-estimated parameter(s) do not ensure the best choice(s).
2023-02-14 14:34:57,361 - INFO: If the result graph is not a circular organelle genome,
2023-02-14 14:34:57,361 - INFO: you could adjust the value(s) of '-w'/'-R' for another new run.
2023-02-14 14:35:06,259 - INFO: Pre-assembling mapped reads ...
2023-02-14 14:35:16,171 - INFO: Pre-assembling mapped reads finished.
2023-02-14 14:35:16,172 - INFO: Estimated embplant_pt-hitting base-coverage = 290.50
2023-02-14 14:35:16,442 - INFO: Estimated word size(s): 112
2023-02-14 14:35:16,443 - INFO: Setting '-w 112'
2023-02-14 14:35:16,443 - INFO: Setting '--max-extending-len inf'
2023-02-14 14:35:16,892 - INFO: Checking seed reads and parameters finished.

2023-02-14 14:35:16,893 - INFO: Making read index ...
2023-02-14 14:38:28,854 - INFO: 26054075 candidates in all 30000000 reads
2023-02-14 14:38:28,854 - INFO: Pre-grouping reads ...
2023-02-14 14:38:28,854 - INFO: Setting '--pre-w 112'
2023-02-14 14:38:31,625 - INFO: 200000/3396434 used/duplicated
2023-02-14 14:38:46,121 - INFO: 4275 groups made.
2023-02-14 14:38:49,639 - INFO: Making read index finished.

2023-02-14 14:38:49,639 - INFO: Extending ...
2023-02-14 14:38:49,639 - INFO: Adding initial words ...
2023-02-14 14:38:54,844 - INFO: AW 1600880
2023-02-14 14:41:13,063 - INFO: Round 1: 26054075/26054075 AI 206553 AW 1970418
2023-02-14 14:43:28,111 - INFO: Round 2: 26054075/26054075 AI 215489 AW 2019166
2023-02-14 14:45:43,049 - INFO: Round 3: 26054075/26054075 AI 216536 AW 2029506
2023-02-14 14:47:56,812 - INFO: Round 4: 26054075/26054075 AI 216561 AW 2030180
2023-02-14 14:50:10,902 - INFO: Round 5: 26054075/26054075 AI 216581 AW 2030482
2023-02-14 14:52:25,998 - INFO: Round 6: 26054075/26054075 AI 216581 AW 2030482
2023-02-14 14:52:25,998 - INFO: No more reads found and terminated ...
2023-02-14 14:52:58,777 - INFO: Extending finished.

2023-02-14 14:52:59,681 - INFO: Separating extended fastq file ...
2023-02-14 14:53:00,424 - INFO: Setting '-k 21,45,65,85,105,127'
2023-02-14 14:53:00,424 - INFO: Assembling using SPAdes ...
2023-02-14 14:53:00,476 - INFO: spades.py -t 20 --phred-offset 33 -1 V1/extended_1_paired.fq -2 V1/extended_2_paired.fq --s1 V1/extended_1_unpaired.fq --s2 V1/extended_2_unpaired.fq -k 21,45,65,85,105,127 -o V1/extended_spades
2023-02-14 14:53:51,072 - INFO: Insert size = 355.059, deviation = 73.8438, left quantile = 264, right quantile = 450
2023-02-14 14:53:51,073 - INFO: Assembling finished.

2023-02-14 14:54:00,154 - INFO: Slimming V1/extended_spades/K127/assembly_graph.fastg finished!
2023-02-14 14:54:00,154 - INFO: Slimming assembly graphs finished.

2023-02-14 14:54:00,154 - INFO: Extracting embplant_pt from the assemblies ...
2023-02-14 14:54:00,155 - INFO: Disentangling V1/extended_spades/K127/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-02-14 14:54:00,503 - INFO: Disentangling failed: 'Incomplete/Complicated graph: please check around EDGE_333!'
2023-02-14 14:54:00,503 - INFO: Scaffolding disconnected contigs using SPAdes scaffolds ...
2023-02-14 14:54:00,503 - WARNING: Assembly based on scaffolding may not be as accurate as the ones directly exported from the assembly graph.
2023-02-14 14:54:00,504 - INFO: Disentangling V1/extended_spades/K127/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-02-14 14:54:00,526 - WARNING: -200-bp gap/overlap between 369 and 351 indicated while conflicting connections existed!
2023-02-14 14:54:00,527 - WARNING: -100-bp gap/overlap between 361 and 4566 indicated while conflicting connections existed!
2023-02-14 14:54:00,783 - INFO: Disentangling failed: 'Incomplete/Complicated graph: please check around EDGE_333!'
2023-02-14 14:54:00,783 - INFO: Disentangling V1/extended_spades/K127/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a/an embplant_pt-insufficient graph ...
2023-02-14 14:54:01,655 - INFO: Vertex_263 #copy = 2
2023-02-14 14:54:01,656 - INFO: Vertex_297 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_327 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_333 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_61 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_291 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_321_4442_253 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_4556 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_4664 #copy = 2
2023-02-14 14:54:01,656 - INFO: Vertex_315 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_367 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_4228 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_4780 #copy = 2
2023-02-14 14:54:01,656 - INFO: Vertex_5022 #copy = 1
2023-02-14 14:54:01,656 - INFO: Vertex_5026 #copy = 2
2023-02-14 14:54:01,656 - INFO: Vertex_7 #copy = 1
2023-02-14 14:54:01,656 - INFO: Average embplant_pt kmer-coverage = 56.3
2023-02-14 14:54:01,656 - INFO: Average embplant_pt base-coverage = 351.7
2023-02-14 14:54:01,656 - INFO: Writing output ...
2023-02-14 14:54:01,712 - WARNING: More than one structure (gene order) produced ...
2023-02-14 14:54:01,713 - WARNING: Please check the final result to confirm whether they are simply different in SSC direction (two flip-flop configurations)!
2023-02-14 14:54:01,714 - INFO: Writing PATH1 of embplant_pt scaffold(s) to V1/embplant_pt.K127.scaffolds.graph1.1.path_sequence.fasta
2023-02-14 14:54:01,716 - INFO: Writing PATH2 of embplant_pt scaffold(s) to V1/embplant_pt.K127.scaffolds.graph1.2.path_sequence.fasta
2023-02-14 14:54:01,716 - INFO: Writing GRAPH to V1/embplant_pt.K127.contigs.graph1.selected_graph.gfa
2023-02-14 14:54:01,716 - INFO: Result status of embplant_pt: 4 scaffold(s)
2023-02-14 14:54:01,872 - INFO: Writing output finished.
2023-02-14 14:54:01,872 - INFO: Please ...
2023-02-14 14:54:01,873 - INFO: load the graph file 'assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg' in K127
2023-02-14 14:54:01,873 - INFO: load the CSV file 'assembly_graph.fastg.extend-embplant_pt-embplant_mt.csv' in K127
2023-02-14 14:54:01,873 - INFO: visualize and confirm the incomplete result in Bandage.
2023-02-14 14:54:01,873 - INFO: If the result is nearly complete,
2023-02-14 14:54:01,873 - INFO: you can also adjust the arguments according to https://github.com/Kinggerm/GetOrganelle/wiki/FAQ#what-should-i-do-with-incomplete-resultbroken-assembly-graph
2023-02-14 14:54:01,873 - 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!
2023-02-14 14:54:01,873 - INFO: Extracting embplant_pt from the assemblies finished.

Total cost 1623.70 s
Thank you!