INCOMPLETE ASSEMBLY

[karthickbrlab]

Hello.
Can you please let me know, whether the graph is complete or not.

i found noisy graph in bandage. THE NODES ARE TOO SHORT : 100-150bp in size. Total size is 2.5MB. (Bandage graph image is atteached below)

How can i proceed now to get circular genome. here is my log file details

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 3.6.15 | packaged by conda-forge | (default, Dec 3 2021, 18:49:41) [GCC 9.4.0]
PLATFORM: Linux ubuntu-desktop 5.19.0-46-generic #47~22.04.1-Ubuntu SMP PREEMPT_DYNAMIC Wed Jun 21 15:35:31 UTC 2 x86_64 x86_64
PYTHON LIBS: GetOrganelleLib 1.7.7.0; numpy 1.19.5; sympy 1.5.1; scipy 1.5.3
DEPENDENCIES: Bowtie2 2.3.5.1; SPAdes 3.13.0; Blast 2.5.0
GETORG_PATH=/home/ubuntu/.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: /home/ubuntu/GetOrganelle
/home/ubuntu/anaconda3/envs/getorganelle/bin/get_organelle_from_reads.py -1 sample_L001_R1.fastq.gz -2 sample_L001_R2.fastq.gz -o plastome -R 15 -k 21,45,65,85,105 -F embplant_pt --reduce-reads-for-coverage inf --max-reads inf

2023-08-01 18:56:57,129 - INFO: Pre-reading fastq ...
2023-08-01 18:56:57,130 - INFO: Unzipping reads file: sample_L001_R1.fastq.gz (460946363 bytes)
2023-08-01 18:57:31,934 - INFO: Unzipping reads file: sample_L001_R2.fastq.gz (666243623 bytes)
2023-08-01 18:57:53,925 - INFO: Counting read qualities ...
2023-08-01 18:57:54,069 - INFO: Identified quality encoding format = Illumina 1.8+
2023-08-01 18:57:54,069 - INFO: Phred offset = 33
2023-08-01 18:57:54,070 - INFO: Trimming bases with qualities (0.00%): 33..33 !
2023-08-01 18:57:54,119 - INFO: Mean error rate = 0.0044
2023-08-01 18:57:54,120 - INFO: Counting read lengths ...
2023-08-01 18:58:17,962 - INFO: Mean = 151.0 bp, maximum = 151 bp.
2023-08-01 18:58:17,962 - INFO: Reads used = 11913168+11913168
2023-08-01 18:58:17,962 - INFO: Pre-reading fastq finished.

2023-08-01 18:58:17,962 - INFO: Making seed reads ...
2023-08-01 18:58:17,962 - INFO: Seed bowtie2 index existed!
2023-08-01 18:58:17,962 - INFO: Mapping reads to seed bowtie2 index ...
2023-08-01 19:34:20,439 - INFO: Mapping finished.
2023-08-01 19:34:20,440 - INFO: Seed reads made: plastome/seed/embplant_pt.initial.fq (2614719682 bytes)
2023-08-01 19:34:20,442 - INFO: Making seed reads finished.

2023-08-01 19:34:20,443 - INFO: Checking seed reads and parameters ...
2023-08-01 19:34:20,443 - INFO: The automatically-estimated parameter(s) do not ensure the best choice(s).
2023-08-01 19:34:20,443 - INFO: If the result graph is not a circular organelle genome,
2023-08-01 19:34:20,443 - INFO: you could adjust the value(s) of '-w'/'-R' for another new run.
2023-08-01 19:38:20,809 - INFO: Pre-assembling mapped reads ...
2023-08-02 22:03:09,488 - INFO: Pre-assembling mapped reads finished.
2023-08-02 22:03:09,495 - INFO: Estimated embplant_pt-hitting base-coverage = 4430.82
2023-08-02 22:03:12,129 - INFO: Estimated word size(s): 113
2023-08-02 22:03:12,129 - INFO: Setting '-w 113'
2023-08-02 22:03:12,129 - INFO: Setting '--max-extending-len inf'
2023-08-02 22:03:32,604 - INFO: Checking seed reads and parameters finished.

2023-08-02 22:03:32,604 - INFO: Making read index ...
2023-08-02 22:06:15,454 - INFO: 22274126 candidates in all 23826336 reads
2023-08-02 22:06:17,396 - INFO: Pre-grouping reads ...
2023-08-02 22:06:17,396 - INFO: Setting '--pre-w 113'
2023-08-02 22:06:18,860 - INFO: 200000/493461 used/duplicated
2023-08-02 22:06:30,893 - INFO: 2173 groups made.
2023-08-02 22:06:33,281 - INFO: Making read index finished.

2023-08-02 22:06:33,281 - INFO: Extending ...
2023-08-02 22:06:33,282 - INFO: Adding initial words ...
2023-08-02 22:11:16,342 - INFO: AW 306937220
2023-08-02 22:16:12,260 - INFO: Round 1: 17150981/22274126 AI 9958129 AW 400076864
2023-08-02 22:16:12,270 - INFO: Hit the words limit and terminated ...
2023-08-02 22:16:12,270 - WARNING: Terminated at an insufficient number of rounds, see '--max-n-words'/'--max-extending-len' for more.
2023-08-02 23:43:08,017 - INFO: Extending finished.

2023-08-02 23:43:09,024 - INFO: Separating extended fastq file ...
2023-08-02 23:43:48,574 - INFO: Setting '-k 21,45,65,85,105'
2023-08-02 23:43:48,574 - INFO: Assembling using SPAdes ...
2023-08-02 23:43:48,685 - INFO: spades.py -t 1 --phred-offset 33 -1 plastome/extended_1_paired.fq -2 plastome/extended_2_paired.fq --s1 plastome/extended_1_unpaired.fq --s2 plastome/extended_2_unpaired.fq -k 21,45,65,85,105 -o plastome/extended_spades
2023-08-03 09:15:25,585 - INFO: Insert size = 151, deviation = 0, left quantile = 151, right quantile = 151
2023-08-03 09:15:25,632 - INFO: Assembling finished.

2023-08-03 09:15:39,987 - INFO: Slimming plastome/extended_spades/K105/assembly_graph.fastg finished!
2023-08-03 09:15:49,661 - INFO: Slimming plastome/extended_spades/K85/assembly_graph.fastg finished!
2023-08-03 11:59:09,418 - INFO: Slimming plastome/extended_spades/K65/assembly_graph.fastg finished!
2023-08-03 12:59:24,188 - INFO: Slimming plastome/extended_spades/K45/assembly_graph.fastg finished!
2023-08-03 12:59:24,234 - INFO: Slimming assembly graphs finished.

2023-08-03 12:59:24,259 - INFO: Extracting embplant_pt from the assemblies ...
2023-08-03 12:59:24,302 - INFO: Disentangling plastome/extended_spades/K105/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-08-03 12:59:25,866 - INFO: Disentangling failed: 'Incomplete/Complicated graph: please check around EDGE_112116512_112124090_112931786_113641422_113641430_113641438_113643106_113643114_113643122_113643130_113643138_113643146_113643154_113643162_113643170_113643178_113643186_113643194_113643202_113643210_114267866_114267864_114267856_114325346_114325354_114325362_114325370_114325378_114328752_114331634_114331642_114331650_114331658_114331666_114331674_114331682_114331690_114331698_114331706_114331714_114331722_114331730_114331738_114331746_114331754_114331762_114331770_114331778_114331786_114331794_114332278_114332286_114332294_114332302_114332310_114332318_114332326_114332334_114332342_114332350_114332358_114332366_114332374_114332382_114332390_114332398_114332406_114332414_114332422_114332430_114332438_114332446_114332454_114332462_114332470_114332478_114332486_114332494_114332502_114332510_114332518_114332526_114332534_114332542_114332550_114332558_114332566_114332574_114332582_114332590_114332598_114332606_114332614_114332622_114332630_114332638_114332646_114332654_114332662_114332670_114332678_114332686_114332694_114332702_114332710_114332718_114332726_114332734_114332266_114332270_114331626_114330008_114330000_114329992_114329984_114329976_114329968_114330452_114327704_114327696_114328164_114323584_114323592_114323600_114323602_114323212_114323216_114315386_114103934_114168252_114312714_114312708_114315750_114315378!'
2023-08-03 12:59:25,868 - INFO: Disentangling plastome/extended_spades/K85/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-08-03 12:59:28,321 - INFO: Disentangling failed: 'Multiple isolated embplant_pt components detected! Broken or contamination?'
2023-08-03 12:59:28,323 - INFO: Disentangling plastome/extended_spades/K65/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-08-03 13:16:39,673 - INFO: Disentangling failed: 'Multiple isolated embplant_pt components detected! Broken or contamination?'
2023-08-03 13:16:40,296 - INFO: Disentangling plastome/extended_spades/K45/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-08-03 13:20:19,383 - INFO: Disentangling failed: 'Multiple isolated embplant_pt components detected! Broken or contamination?'
2023-08-03 13:20:19,656 - INFO: Scaffolding disconnected contigs using SPAdes scaffolds ...
2023-08-03 13:20:19,656 - WARNING: Assembly based on scaffolding may not be as accurate as the ones directly exported from the assembly graph.
2023-08-03 13:20:19,656 - INFO: Disentangling plastome/extended_spades/K105/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2023-08-03 13:20:19,881 - INFO: Disentangling failed: 'No new connections.'
2023-08-03 13:20:19,885 - INFO: Disentangling plastome/extended_spades/K105/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a/an embplant_pt-insufficient graph ...
2023-08-03 13:20:20,553 - INFO: Average embplant_pt kmer-coverage = 24409.2
2023-08-03 13:20:20,553 - INFO: Average embplant_pt base-coverage = 78424.3
2023-08-03 13:20:20,553 - INFO: Writing output ...
2023-08-03 13:20:20,553 - INFO: Writing PATH1 of embplant_pt scaffold(s) to plastome/embplant_pt.K105.scaffolds.graph1.1.path_sequence.fasta
2023-08-03 13:20:20,570 - INFO: Writing GRAPH to plastome/embplant_pt.K105.contigs.graph1.selected_graph.gfa
2023-08-03 13:20:20,570 - INFO: Result status of embplant_pt: 1 scaffold(s)
2023-08-03 13:20:20,796 - INFO: Writing output finished.
2023-08-03 13:20:20,814 - INFO: Please ...
2023-08-03 13:20:20,814 - INFO: load the graph file 'assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg' in K105,K85,K65,K45
2023-08-03 13:20:20,814 - INFO: load the CSV file 'assembly_graph.fastg.extend-embplant_pt-embplant_mt.csv' in K105,K85,K65,K45
2023-08-03 13:20:20,814 - INFO: visualize and confirm the incomplete result in Bandage.
2023-08-03 13:20:20,814 - INFO: If the result is nearly complete,
2023-08-03 13:20:20,814 - 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
2023-08-03 13:20:20,814 - 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!
2023-08-03 13:20:20,814 - INFO: Extracting embplant_pt from the assemblies finished.

HERE I UPLOAD THE ASSEMBLY GRAPH--

[JianjunJin]

Given the extremely high depth, you should have the sufficient graph.
However, it's hard to tell from such complex graph. You may try to constraint the depth range in Bandage to remove the potential contaminants or mtpt.

[karthickbrlab]

Can i use any reference genome and use join_spades_by_blast.py ?

[JianjunJin]

I apologize for overlooking your thread; it was an unintentional slip due to other commitments.

As previously mentioned, the complete plastome is likely contained within the complex graph. To isolate the plastid, you can filter out contigs with lower depth and those not part of the plastome. Utilize the --min-depth parameter in slim_graph.py along with manual curation using Bandage. Should you require additional assistance, please feel free to reach out.