Help needed to polish pacbiotoca assembly with Quiver

[cwzkevin]

Dear PacBio team,

I have used CA8.1 to assemble pacbio self-corrected sequences. In the wiki of PacBioToCA, it has such "After assembly, polishing with Quiver is recommended to get the final consensus sequence".

In my system, I don't have and can't have SmrtAnalysis or SmrtPortal installed. I installed each program that needed through the progress. For example, I installed BLASR when CA8.1 self-correction needed it. And, I installed Quiver just now.

However, in HowToQuiver, I can't follow Step 3 or Step 4. For step3, since I don't have aligned_reads.cmp.h5, I was trying to find out how to follow the RS_Mapping_QVs protocol. But when I google "RS_Mapping_QVs protocol", all I can get is HowToQuiver.rst. I am in a dead end. For step4, since I don't have and can't have SmrtPortal, I am also in a dead end.

Can you please help me? It seems to me that as long as I can create the aligned_reads.cmp.h5 file, then, I am good to run Quiver. Thanks!

[GenoMax]

See if thread helps: http://seqanswers.com/forums/showthread.php?t=35212

[cwzkevin]

See if thread helps: http://seqanswers.com/forums/showthread.php?t=35212

Thanks. Now, I installed pbalign. The following command caused error.

pbalign.py --forQuiver input_20131217_RS42150_PL100038726-1_D02.1.bas.h5 reference.fna aligned_reads.cmp.h5

2014-01-12 20:41:22,148 [ERROR] BlaserService returned a non-zero exit status. [INFO] 2014-01-12T20:41:21 [blasr] started.
ERROR, ccs base file input_20131217_RS42150_PL100038726-1_D02.1.bas.h5 does not have a PulseData field.
Traceback (most recent call last):
  File "/home/qiime/VE-QUIVER/bin/variantCaller.py", line 5, in <module>
    sys.exit(main())
  File "/home/qiime/VE-QUIVER/lib/python2.7/site-packages/GenomicConsensus/main.py", line 340, in main
    return ToolRunner().main()
  File "/home/qiime/VE-QUIVER/lib/python2.7/site-packages/GenomicConsensus/main.py", line 265, in main
    with CmpH5Reader(options.inputFilename) as peekCmpH5:
  File "/home/qiime/VE-QUIVER/lib/python2.7/site-packages/pbcore/io/CmpH5IO.py", line 716, in __init__
    self.file = h5py.File(self.filename, "r")
  File "/home/qiime/lib/python/h5py/_hl/files.py", line 207, in __init__
    fid = make_fid(name, mode, userblock_size, fapl)
  File "/home/qiime/lib/python/h5py/_hl/files.py", line 79, in make_fid
    fid = h5f.open(name, h5f.ACC_RDONLY, fapl=fapl)
  File "h5f.pyx", line 71, in h5py.h5f.open (h5py/h5f.c:1682)
IOError: unable to open file (File accessibilty: Unable to open file)

What should I do? Thank you!

[rhall]

Is the data from an RSII instrument? In which case pbalign requires the bax.h5 files, not the bas.h5 file. To use all three bax.h5 files simply create a fofn of the bax.h5 file.

ls *.bax.h5 > input.fofn

and pass the input.fofn file to pbalign

pbalign.py --forQuiver input.fofn reference.fna aligned_reads.cmp.h5

[cwzkevin]

Thank you, rhall. It got another error.

2014-01-13 21:25:35,665 [ERROR] FilterService returned a non-zero exit status. /bin/sh: samFilter: command not found

Never mind, I found them in blasr/pbihdfutils/bin

Thanks for your help!