Reg: Mitochondrial Genome Assembly about getorganelle HOT 8 CLOSED

Hi!

Generally, for assembling any kind of organelle genome, you have to firstly get the organelle-sufficient graph, which I defined to be an assembly graph with contigs completely covers the whole organelle genome. In your case, the assembly graph is not sufficient, meaning that there are about three gaps. Considering the k-mer coverage, this is nice data and it is very likely to get the mitogenome-sufficient graph after adjusting some options when running GetOrganelle. Could you please attach the get_org.log.txt file? I can barely tell what to do to improve the assembly graph.

Only when we did obtain the mitogenome-sufficient graph, we could disentangle the graph into complete sequences. You actually do not need to watch the video and manually disentangle a nice graph like this after you make it "sufficient". You can simply use the script disentangle_organelle_assembly.py (which would be automatically called by the main script). However, for mitogenome with large scale repeats like this graph, there would be lots possible paths, which are potential configurations for the mitogenome. Even if you had really long insert size for the sequencing library, different isomers of mitogenome (even of plastome) coexist in plant is quite common.

Let's look at the log file first~

from getorganelle.

Alright! I'm attaching the log-file! I went through the video while going through the paper and I thought it showed good clarity!

get_org.log.txt

Please do let me know, currently I have just used the default parameters with the anti-seed being set as a chloroplast genome

from getorganelle.

I would try using a broader range of k-mer gradient, "-k 21,35,45,55,75,95,105,115,127" first.
You could make a duplicate for previous result and deleted the filtered_spades folder and re-run get_organelle_reads.py with your previous options together with "-k 21,35,45,55,75,95,105,115,127 --continue", thus the previous extension part would be skipped.

Let me know the progress!

from getorganelle.

Sure, I'm currently starting the run again.

from getorganelle.

Hi,

I have finished the run for the same sample using the extended kmer gradient as per your suggestion, I'm also attaching the log file.

It seems that the graph is still mitogenome-insufficient and as such can't be circularized.

Thanks for the help
get_org.log.txt

from getorganelle.

Hi,

I mean you should try "-k 21,35,45,55,75,95,105,115,127" rather than 75,85,95,105,115,127 in your log file. Also, you have to delete previous assembly_graph.fastg files in the output folder, otherwise the slim & disentangle process would be skipped as well when "--continue" used.
If you cannot quite get the usage of continue, you can start a new run by using:

get_organelle_reads.py -1 ../../1_R1.clean.fastq -2 ../../1_R2.clean.fastq -s /data/ref_genomes/Eucalyptus_grandis/Eucalyptus_mitochondrion.fasta -a /data/ref_genomes/pomogranate/chloroplast.fasta --genes /data/ref_genomes/Eucalyptus_grandis/Eucalptus_MT_coding_sequnce.fasta -o mitochondria_Sample1 -R 30 -t 20 -k 21,35,45,55,75,95,105,115,127 -F plant_mt

If by doing this you still fail with using "-k 21,35,45,55,75,95,105,115,127", you may try to remove the "-a /data/ref_genomes/pomogranate/chloroplast.fasta" option, for the target mitogenome might share some chloroplast gene/sequence and was removed by the anti-seed. But I would try the "-k 21,35,45,55,75,95,105,115,127" first.

from getorganelle.

Thank you, I'll do that and get back to you.

from getorganelle.

Since there are no follow-ups, I'm closing this issue. Feel free to reopen it if you have further questions.

from getorganelle.