DeNovo assembly using pacBio data

[jbingham]

Your best bet will be to use the FASTQ files rather than the raw bas.h5 files. You can download them from the E coli page here:

http://www.pacbiodevnet.com/Share/Datasets/E-coli-Outbreak

For example, you could grab the two filtered subread files for C227-11: one is for CCS, the other for long reads.

Note that you could download the error-corrected version of the reads as a FASTQ from the same page. To do the error correction yourself, your best bet is pacBioToCA and the Celera Assembler. There are links to them both here:

http://www.pacbiodevnet.com/CodeShare_Project?id=a1q70000000GrT6AAK

One reason is that PacBio's error correction pipeline will be incorporated in the next software release.

Also, Mike Schatz's presentation is really useful:

http://schatzlab.cshl.edu/presentations/2011-09-07.PacBio%20Users%20Meeting.pdf

[krittika.sasmal]

Pipeline for Corrected Long read generation

Thank you for all your answers. Can anybody tell me the pipeline to be followed to generate ths error corrected CLR reads. I have downloaded the SMRT pipe.
Moreover how are the filtered reads generated? Help me out with the SMRT analysis. I downloaded E.coli raw reads from DevNet. However there seem to be several bas.h5 files. Do I combine them and proceed.
What are the parameters BLASR takes. Can anybody help?

[jbingham]

PacBio's base caller outputs sequence data in HDF5 format, PacBio's native data format. The HDF files contain base calls for both long reads and circular consensus (if applicable, meaning the reads wrapped around the adapters), as well as quality scores and kinetic measurements. PacBio provides APIs in Python, R and Java for accessing the files. You can download them from www.pacbiodevnet.com.

When using PacBio's secondary analysis pipeline, you'll get alignments in SAM/BAM+BAI, coverage in BED and GFF, variant calls in GFF and VCF, as well as the FASTA/FASTQ for filtered subreads.

[krobison]

MIRA and Celera Assembler are two other assemblers which support de novo assembly using PacBio, and perhaps more importantly mixing PacBio with other technologies.

[SillyPoint]

There's a high-level how-to on pacbiodevnet, describing how they used a combination of consensus and long reads to do de novo assembly of an E.coli strain.

As I understand it, the consensus reads are generated during the primary analysis step, which occurs on the PacBio server. The main product of that step is the *.bas.h5 file, which includes basecalls, several quality scores, and limited kinetics info. At this point, adapters have been identified, and reads can be split into sub-reads.

The secondary analysis, running on your cluster, does filtering of subreads based on productivity, high-quality region length and score, to produce a filtered_subreads.fasta file. It also removes control reads (although those are still prersent in filtered_subreads.fasta -- I think), and then performs an alignment to the reference provided in the protocol to produce a BAM file and various other stuff.

That's my view-from-40000-feet, anyway. Perhaps a helpful PacBio person will wander by and provide a bit more detail.

[krittika.sasmal]

Hi,
I am new to PacBio. I have visited the pacBio site. However could not clearly understand what files are generated by the primary analysis or base calling. Is it the CLR or CCS files? how do we actually go about generating the filtered read files. If I am interested to use the SMRT pipe, what input does it take?

Also, can anybody please suggest a de novo assembler that works well with PacBio data?