Question: PacBio assembly - pbalign error
0
gravatar for hokeak
8 months ago by
hokeak0
hokeak0 wrote:

I am assembling PacBio sequencing data for five different bacterial isolates. I ran the sequence files through Canu and now I am trying to run them through pbalign so I can run them through Quiver. I finally got pbalign to run but it comes back with "Error: can not convert non-pacbio reads to pbbam record." I have absolutely no idea what I am doing wrong. If anyone has any experience with PacBio assembly I would love some input!

pacbio assembly genome • 553 views
ADD COMMENTlink written 8 months ago by hokeak0

Can you post the command you are trying to execute so we can have a look?

So you try to align the original pacbio reads to the assembled ones, right?

ADD REPLYlink written 8 months ago by lieven.sterck5.5k

Basically: $ pbalign assembly.fasta reference.fasta alignment.bam. I also tried to just run it through blasr and got the same error. What Google seems to be telling me is that the assembly is not being recognized as pacbio format?

ADD REPLYlink written 8 months ago by hokeak0

I'm not sure but I think you might be confused about the purpose of pbalign

pbalign is used to align raw (as in: un-assembled) pacbio reads to a reference , not to align a canu assembly to a reference. Moreover pbalign outputs in sam format, not bam , so unless you plan to seriously confuse yourself I suggest renaming the output file ;)

ADD REPLYlink modified 8 months ago • written 8 months ago by lieven.sterck5.5k

You are correct, I am incredibly confused because when I ran Quiver it asked if they'd been aligned so maybe I'll try Arrow? I basically have no idea what I am doing and everyone I know uses Illumina. Thanks for your help!

ADD REPLYlink written 8 months ago by hokeak0

What I think you have to do is to align your raw pacbio reads to the assembly you created from it.

pbalign raw_pbreads.fasta assembled.fasta output.sam

the result of that should be ok as input for Quiver

ADD REPLYlink written 8 months ago by lieven.sterck5.5k

Unfortunately I still get the same error :/

ADD REPLYlink written 8 months ago by hokeak0

Can you post a few header lines of you raw_pacbio read file(s)? Only a few lines starting with a '>' will do .

Did you had a look at the manual for pbalign btw? I read this in it:

example (2.3) - Create a cmp.h5 file with --forQuiver option

# The output cmp.h5 file will loaded with quality values (pulses)
# from the input bas/bax.h5 file, sorted and repacked, and therefore
# can be consumed by Quiver directly, (Note that in order to use
# --forQuiver option, cmph5tools and h5repack are required.)

$ pbalign --forQuiver your_movie.bas.h5 your_reference.fasta out.cmp.h5

so, you will need the bas.h5 file as input for pbalign (not the fasta thus apparently), but this is not related to your error I assume

ADD REPLYlink modified 8 months ago • written 8 months ago by lieven.sterck5.5k
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