result from the initial mapping assembly is al XXXXX

[regina.lo...@gmail.com]

Hi Christoph,

I ran the --quick version with the --verbose option. The temp_baitfile.fasta file from the iteration0 seems to be ok (18,415bp). The output of the initial-mapping-testpool-to-perna-mt_out_testpool.unpadded.fasta is not ok (I copied below a part of it but it is al xxx's till the end).
See in the attached file the output of the run with the --quick and --verbose option.

Many thanks,
Regina

>KM655841.1_bb
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
[….]




On 05 Dec 2016, at 13:21, Christoph Hahn <C.H...@hull.ac.uk> wrote:

Hi Regina,

That's indeed a bit strange. If you are using a mt genome of the same species, you won't need the initial mapping assembly. You might as well just use the `--quick` option. Anyway, now that you've already done it, can you please double-check that a bait file has been created and maybe look into it to check if it contains a reasonable sequence. The file should be called `temp_baitfile.fasta` in your iteration 1 directory. This is the fasta sequence (without ambiguities) extracted from the result (maf file) of the initial assembly. Could be something has gone wrong already there. Maybe the file is empty or otherwise weird, that would explain why no reads seem to be baited. Check out also the result from the initial mapping assembly. Should be in the directory called `your project_d_results` and the file should be called `your_project_out.unpadded.fasta` or something like that.
In parallel can you try to rerun MITObim with mira 4.0.2 and the `--quick` option? I know you already tried, but the this time please specify the verbose (`-v`), which should result in more detailed output from the baiting step. Can you save the output to a txt file and send it over, in case it fails?

Thanks!

Cheers,
Christoph

[Chris H]

Hi,

Thanks! 

Sorry, I am not sure if we are talking about the same thing now. The `temp_baitfile.fasta` in iteration0 is just a copy of the reference that you provide via `--quick`. According to the log you sent no reads seem to be baited, which is strange. Could you just clarify a few things for me please: 

- In your initial message you wrote that you successfully completed a mapping assembly, first. Did you get a sensible result from this mapping assembly or is the result the file containing all Xs that you mentioned? 

- Did you specify to MIRA and/or MITObim that you are processing Iontorrent data?

Before, we go any further, could you please also try to run tutorial II with the testdata that comes with MITObim. Just to make sure everyhing is running fine in principle. Sorry for the inconvenience.

cheers,

Christoph 

[regina.lo...@gmail.com]

Hi Christoph,

thanks for your reply. Yes, this is about a previous post.
I just ran Tutorial II with the testdata as you requested and everything is ok.

I decided to run the quick approach with another reference (same genus, different species) because I checked the one I was using from the same species and has long intergenic spaces that might be causing problems.
Results remain disappointing.
I am attaching the command-lines I used and the results. The initial mapping is already not good (file attached)

Could it be a problem with the raw data? The report the company sent about this sample indicated the following:
Sample ID N bases [Mbp]   N reads  Mean length [bp]
   PC                54,2             228 710        237


I specified to MIRA that I'm processing iontorrent
#manifest file for basic mapping assembly with IonTorrent data using MIRA 4

project = initial-mapping-testpool-to-PC-mt

job=genome,mapping,accurate

parameters = -NW:mrnl=0 -AS:nop=1 IONTOR_SETTINGS -CO:msr=no

readgroup
is_reference
data = /home/regina/mtDNA/viridismtDNA.fa
strain = PC-mt-genome

readgroup = reads
data = /home/regina/mtDNA/PC.fastq
technology = iontor
strain = testpool


Any idea of why I'm getting so bad results?

Thanks
Regina

[drur...@gmail.com]

Hi, I just posted to the older question then realized the question was moved to here, so this is just the same question. I am trying to use MITObim with mira and had the same issue of using the wrong verion, 4.9.x. I am new to ubuntu and I've tried finding this on my own, but I can't seem to figure it out. How do I get mira version 4.0.2? I tried this

sudo apt-get purge mira-assembler

which seemed to remove it successfully, but when I tried to install 4.0.2, like this

sudo apt-get install mira-assembler=4.0.2

I received the error:

E: Version '4.0.2' for 'mira-assembler' was not found

I tried finding any extension that may need to go on that version, but I couldn't find anything that worked. Do you have any suggestion how to do this?

Thanks,

Austin

[Chris H]

Hello Austin,

Not sure why you thought your question is related to the problem Regina was having. Next time please consider to start a new post if you have a question that is not directly related to an ongoing discussion. I suggested a solution to your problem in the other post. Thanks! cheers,
Christoph

[Chris H]

Dear Regina,

Really sorry for the long delay. Swamped with a million things. Did you have any success figuring out what could be going wrong there? I was just thinking if you might just have very low coverage. Have you ever looked at the result from the assembly with an appropriate viewer? How's the coverage in the few parts where you do get an assembly?

cheers,
Christoph