An incomplete cp genome assembly.

[SYSU-SinLee]

Hi, I'm trying to assemble the chloroplast genome of one dicotyledon plant.

First, I ran GetOrganelle with the parameters -F embplant_pt -R 10 -t 1 -k 21,45,65,85,105, and got an incomplete assembly (the log file, gfa and fastg files are attached with the prefix "run1").

Then I increased the number of rounds to run and added a reference cp genome from a close related species with parameters -F embplant_pt -o plastome -R 20 -t 10 -k 21,45,65,85,105 -s ../Ref.fa. The results look better but are still incomplete (files with the prefix "run2").

To improve the completeness of the assembly, I increased the round to run again and also increased the disentangle time limit with parameters -F embplant_pt -o plastome -R 40 -t 10 -k 21,45,65,85,105 -s ../Ref.fa --disentangle-time-limit 72000. But the assembly is not circular (files with the prefix "run3" and the figures shown below).

Now I'm trying to run GetOrganelle with an increased value of "-w". And I'll appreciate it if there are any suggestions for this problem. Thanks.

Best regards,
Sin Lee

[SYSU-SinLee]

To make it clear and in case it is inconvenient to download and decompress, here are the figures and log files.

The first run:
-F embplant_pt -R 10 -t 1 -k 21,45,65,85,105


run1.log.txt

The second run:
-F embplant_pt -o plastome -R 20 -t 10 -k 21,45,65,85,105 -s ../Ref.fa


run2.log.txt

The third run:
-F embplant_pt -o plastome -R 40 -t 10 -k 21,45,65,85,105 -s ../Ref.fa --disentangle-time-limit 72000

run3.log.txt

Thanks for your time.

[SYSU-SinLee]

Here are the results with -w 95.
-F embplant_pt -o plastome -R 40 -t 10 -k 21,45,65,85,105 -s ../Ref.fa --disentangle-time-limit 72000 -w 95

GFA graph:

Fastg graph:

Log:
run7.log.txt

[Kinggerm]

Please follow the guidelines here: https://github.com/Kinggerm/GetOrganelle/wiki/FAQ#what-should-i-do-with-incomplete-resultbroken-assembly-graph. The top-recommended options are not tried or misunderstood.

1. The top option was reducing the word size, but you were increasing the word size from the automatically-estimated 73 to 95, which worsened the result.
2. You have 20Gb reads, but all the runs were using 2.37 Gb reads, see --reduce-reads-for-coverage or --max-reads in https://github.com/Kinggerm/GetOrganelle/wiki/Usage#main-scripts or through get_organelle_from_reads.py --help.

Next time, please turn on the depths and lengths of contigs in Bandage. Also, I recommend only uploading the *.png rather than the *.gfa or *.fastg files to protect your data privacy.

[SYSU-SinLee]

Thanks for your time. I've followed your suggestions and tried to adjust the parameters as follows.
-R 40 -t 5 -k 21,45,65,85 -s ../REF.fa --disentangle-time-limit 72000 -w 60 --max-reads inf --reduce-reads-for-coverage inf --max-extending-len 6000
Now it seems the assembly is much better.
run11.log.txt

[Kinggerm]

Congrats! Now you have the complete circular genome.

BTW, --max-reads inf --reduce-reads-for-coverage inf is plugging too much data into GetOrganelle and drastically wasting computational time. The time can be much improved to be shorter by picking a number between the default and the infinite.

[SYSU-SinLee]

Hi, I’m trying to assemble the chloroplast genome of a close related species with similar parameters except --reduce-reads-for-coverage 500 as suggested by the manual. Then I found only a very small fraction of the reads were used and the assembly was highly fragmented. Then I tried a series of coverage settings, but the assemblies are all nearly circular with a breakpoint, even when I used all the reads. I have no idea if I should continue to reduce the word size or increase rounds because it will cost such a long time. And I don't know if the reference genome really helps because it's not mentioned where it was used by GetOrganelle in the log file. I‘ll appreciate it if you have any suggestions. Thanks for your time.

For example, when 17.2 million reads were used. (There graph of .fastg is too complicated to show names and lengths on it.)


run9.get_org.log.txt

When all the 380 million reads were used.


run2.log.txt

[Kinggerm]

These two graphs are NOT organelle-sufficient. I recommend finetuning the parameters further following https://github.com/Kinggerm/GetOrganelle/wiki/FAQ#what-should-i-do-with-incomplete-resultbroken-assembly-graph