ERROR while using PBcR

Question

[Medhat]

I am trying to do the example in this link.

the lambda example, I use this command:

wgs-8.2alpha/Linux-amd64/bin/PBcR -length 500 \
  -partitions 200 \
  -l lambdaIll \
  -s pacbio.spec \
  -fastq pacbio.filtered_subreads.fastq \
  genomeSize=50000 \
  illumina.frg

but it gives me this error:

Running with 40.01948X (for genome size 50000) of lambdaIll sequences (2000974 bp).
Correcting with 50X sequences (2500000 bp).
AMOS or PBDAGCON is required to call consensus and neither was available. Please add either SMRTportal or AMOS to your path and try again

I already have pbdagcon in my path.

Any Idea

Thanks in advance

Update: after installing AMOS It worked

[skymningen]

I had similar problems and went through a lot of searching, reinstalling AMOS and such without being able to solve it. In the end I decided to use falcon_sense by adding the -pbCNS flag and figured it was maybe some problem specific to server configuration. Now I think this might really be a bug in PBcR / SMRTanalysis.

Answer 1

[Medhat]

Right now I am installing AMOS and its dependency and I will see what will happens and your suggestion is to use -pbCNS flag ?!

Update: After installing AMOS It worked

Answer 2

[Charles Warden]

I've also been recently been using PBcR with the Celera Assembler. I also found installation of AMOS helped solve a lot of my dependency problems.

FYI, I don't think you should use the -pbCNS flag because it looks like you trying to perform a hybrid assembly.

Here are some pointers I got from the developer, which I found helpful. For example, I learned that the self-correction outperforms the hybrid-correction for "high-coverage" (>200X) assemblies. Given the relatively small size of your genome, this may also be true in your case. So, it is probably useful to perform the following benchmark.

1. Non-hybrid

   Select longest 200X longest pacbio and run PBcR without providing any Illumina data, setting genome size to [genome size] (using genomeSize parameter) and fast consensus (-pbCNS parameter). This will map all the 200X of data to the longest 50X to generate the polished sequences.

   After the correction, take longest 25X of the corrected reads and assemble

2. Hybrid

   Select 50X longest pacbio and 50X illumina and run PBcR with the genomeSize=[genome size] but not the -pbCNS parameter (it doesn't work on Illumina data).

   Again, assemble the longest 25X of the corrected reads

Remember, coverage = (avg read length * number of reads) / genome size

[Medhat]

ERROR: Failed with signal HUP (1)
================================================================================

runCA failed.

----------------------------------------
Stack trace:

 at /home/medhat/source/wgs_pbcr/wgs-8.2alpha/Linux-amd64/bin/runCA line 1568.
    main::caFailure("gatekeeper failed", "/data/assembleBacteria/temppseudo/asm.gkpStore.err") called at /home/medhat/source/wgs_pbcr/wgs-8.2alpha/Linux-amd64/bin/runCA line 1897
    main::preoverlap("/data/assembleBacteria/illumina.frg", "/data/assembleBacteria//temppseudo/pseudo.frg") called at /home/medhat/source/wgs_pbcr/wgs-8.2alpha/Linux-amd64/bin/runCA line 6470

----------------------------------------

Last few lines of the relevant log file (/data/assembleBacteria/temppseudo/asm.gkpStore.err):

Starting file '/data/assembleBacteria/illumina.frg'.

Processing INNIE ILLUMINA 1.3+ QV encoding reads from:
      '/data/assembleBacteria/illumina/SRR491287_1.fastq'
  and '/data/assembleBacteria/illumina/SRR491287_2.fastq'

Starting file '/data/assembleBacteria//temppseudo/pseudo.frg'.

Processing SINGLE-ENDED SANGER QV encoding reads from:
      '/data/assembleBacteria/./pacbio/SRR1042836_1.fastq'


GKP finished with 39443060 alerts or errors:
39443059    # ILL Alert: invalid QV in read.
1    # LIB Alert: suspicious mean and standard deviation; reset stddev to 0.10 * mean.

ERROR: library IID 1 'MATED' has 98.58% errors or warnings.

----------------------------------------

Failure message:

gatekeeper failed

[Charles Warden]

I think you can contact the developers if you have problems running the program: http://www.cbcb.umd.edu/software/pbcr/

It looks like these particular errors come from the Celera Assembler and not PBcR.

Also, I don't see the same text in the comment, but I thought I saw something about "requested PBDAGON but either BLASR or pbdagcon executables were not found" in the original link. I think installing BLASR was helpful, but I was able to get a resutl without actually installing pbdagcon.