Import raw PacBio data from *.bax.h5 files

[reubennowell]

Dear all,

I've been sent some raw PacBio data from a collaborator which I'd like to import into SMRT Portal to generate filtered subreads and CCSs for scaffolding. However I'm struggling to understand the file structure for these data, in terms of what is needed for import into SMRT (and quite possibly PacBio data in general).

A description of the raw data: For each cell, I have three *.bax.h5 files, three *.subreads.fastq files and one *.metadata.xml. There is no *.bas.h5 file... I'm not sure why not. There is also a directory "error corrected" containing a corrected.fastq and a filtered_subreads.fastq.

I'm aware that SMRT requires a certain file structure (e.g., described in this link), and that the bas.h5 file is a pointer to the bax.h5 files. I'd like to know if it's possible to import these data into the SMRT software without the bas.h5, or if it's possible to generate a bas.h5 file from the files I do have?

I'm also a little unsure as to how the sequence files corrected.fastq and filtered_subreads.fastq have been generated - I am guessing that filtered_subreads.fastq contains the adapter-trimmed subreads (duh!), and that corrected.fastq contains the CCS sequences, but is there a way to know for certain?

Any insights will be greatly appreciated!

[ymc]

I think there should be at least one bas.h5 generated by the machine. But this file probably is not necessary at all to run any kind of analysis for the current gen of chemistry. I only need the three bax.h5 file to run the analysis I needed so far.

Hope this helps.

[GenoMax]

@reubennowell: Have you tried to import the SMRTcell(s) into the Portal? It apears that the data has already been partly analyzed.

Link for Wiki for SMRTportal: http://files.pacb.com/software/smrta...MRT_Portal.htm You can find information about various protocols in navigation pane on left.

[reubennowell]

Thanks for the replies both.

@GenoMax, yes I tried to import and received a "no SMRT cell data here" message from the Import SMRT Cells page. I agree this data appears to have been partly analysed; rather than trying to forensically determine what has been done I'd rather just reimport and reanalyse myself.

But I did get it imported in the end All that was required was to restructure the data like this:

data
|---Analysis_Results
|   |---*1.bax.h5
|   |---*2.bax.h5
|   |---*3.bax.h5
|   |---*1.bax.h5
|   |---*2.bax.h5
|   |---*3.bax.h5
|   etc.
|--- *.metadata.xml
|--- *.metadata.xml
etc.

In other words, mkdir Analysis_Results and then mv the bax.h5 files into there - the lack of a bas.h5 file didn't seem to be an issue.

As a side question, are there alternative programs apart from the SMRT software that allow you to go from a bax.h5 to filtered, trimmed and condensed (ie CCS) fastq?

Many thanks again.

[rhall]

The SMRT software is by far the easiest way to generate filtered data, and the only way to generate CCS reads, but a lot of the components are developed individually and are available as development releases on github, e.g. https://github.com/PacificBiosciences/pbcore