Smrtportal/smrtanalysis is the software developed by Pacific Biosciences
We’ll use data from a single smrtcell based on a size selected 20kb E. coli library
Do:
cd /opt/smrtanalysis/common/inputs_dropbox
wget http://files.pacb.com/datasets/secondary-analysis/ecoli-k12-P4C2-20KSS/ecoliK12.tar.gz
tar -vxzf ecoliK12.tar.gz
To import into smrtportal:
Monitor the run, it will probably go overnight
Where is the data
Check /opt/smrtanalysis/common/jobs/016/016XXX, where 016XXX is the job ID from smrtportal. Results appear in the data folder
Use:
See this part of the Pacific Biosciences wiki: https://github.com/PacificBiosciences/SMRT-Analysis/wiki/SMRT-Pipe-Reference-Guide-v2.0#-using-the-command-line
Do:
. /opt/smrtanalysis/etc/setup.sh
fofnToSmrtpipeInput.py input.fofn > input.xml
smrtpipe.py --params=settings.xml xml:input.xml
Or customise smrtpipe to use more cpus (if available):
smrtpipe.py -D NPROC=24 --params=settings.xml xml:input.xml
Results appear in the data folder
Do:
. /opt/smrtanalysis/etc/setup.sh
blasr /opt/smrtanalysis/common/inputs_dropbox/ecoliK12/Analysis_Results/m130404_014004_sidney_c100506902550000001823076808221337_s1_p0.2.bax.h5 \
/path/to/velvet_pe+mp.fa \
-minSubreadLength 1000 -bestn 1 -nproc 8 -sam -out pacbio_2_velvet_pe+mp_71.sam
samtools view -buS pacbio_2_velvet_pe+mp_71.sam | samtools sort - pacbio_2_velvet_pe+mp_71.sorted
samtools index pacbio_2_velvet_pe+mp_71.sorted
The bam and bai files can be added to the IGV browser
Check out https://github.com/PacificBiosciences/Bioinformatics-Training/wiki/Bridgemapper
First, fix a ‘bug’ which makes one of the steps single-cpu instead of parallel.
Edit /opt/smrtanalysis/analysis/lib/python2.7/pbbridgemapper/bridgemapper.py: change lines 426 + 427 from:
stdout, stderr = runBlasr(affixesFastq, args.referenceFasta,
affixBlasrOutputPath)
To:
stdout, stderr = runBlasr(affixesFastq, args.referenceFasta,
affixBlasrOutputPath, nproc=args.nproc)
You can now run Bridgemapper through the smrtportal, which gives the optimal output for viewing in the PacBio genome browser SMRTview. However, this will take a long time. To do that, add the velvet assembly as reference through ‘Home –> Import and Manage –> Manage Reference Sequences –> New’
Alternatively, run bridgemapper on a subset of the reads through the command line. Use screen!
Do:
. /opt/smrtanalysis/etc/setup.sh
pbbridgemapper --debug --nproc 8 /opt/smrtanalysis/common/inputs_dropbox/ecoliK12/Analysis_Results/m130404_014004_sidney_c100506902550000001823076808221337_s1_p0.2.bax.h5 \
/path/to/velvet_pe+mp.fa bridgemapper_out
To use SMRTview with the results, you’ll need to make a smrtportal reference repo:
referenceUploader -c -p smrtpipe_references -n velvet_pe+mp -f /path/to/velvet_pe+mp.fa --saw='sawriter -welter'
Viewing with SMRTView
Start browsing!
Development and posting of this material, and the associated workshop, were supported by Grant Number R25HG006243 from the National Human Genome Research Institute and an NSF OCI supplement to NSF DBI-0939454.
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