CCS

The sm-analysis program uses the ccs program to obtain highly accurate reads from the input BAM that are taken as the true sequences and positions within the reference for each molecule. ccs must be accessible at runtime and it is called on demand: if its output is already there, it will not be computed again.

By default, ccs is searched for in the PATH. If it is not found in the PATH, you will receive a common runtime error message:

[CRITICAL] [Errno 2] No such file or directory: 'ccs'

and the program will terminate.

In that case, the instructions in the following sections can help you.

Installing CCS

Probably the easiest way to install ccs is described in PacBio & Bioconda. The instructions are in summary:

  1. Install conda (see installing conda),

  2. Setup the channels (as described in bioconda channels), and

  3. Install pbccs:

    conda install -c bioconda pbccs
    

Upon success, you will be able to pass the path to the ccs executable to sm-analysis if needed (see below for details).

Using ccs from sm-analysis

Let us assume that PacBio Data Processing was installed inside a virtual environment located in:

/home/david/.venvs/pdp

and let us assume that pbbioconda was installed in:

/home/david/miniconda3

then, after activating the PacBio Data Processing’s virtual environment:

source /home/david/.venvs/pdp/bin/activate

you can tell sm-analysis about ccs by using a command line option (-c/--ccs-path) as follows:

sm-analysis --ccs-path /home/david/miniconda3/bin/ccs

Issues

Multimapping

In some cases an aligned CCS file presents multimapping. Two examples take from the st1A09 file:

m54099_200720_153206/4194505/ccs        0       U00096.3        392180  0       150=    *       0       0       ATCTGTACGTAAGTACGTGATGTCTCCTGCCCACTTCT...
m54099_200720_153206/4194505/ccs        256     U00096.3        1094716 0       150=    *       0       0       ATCTGTACGTAAGTACGTGATGTCTCCTGCCCACTTCT...
m54099_200720_153206/4194505/ccs        272     U00096.3        2170808 0       150=    *       0       0       GGACTGAGGGCAAAGGCCTCCCGGAAGTTCAGCCCGGT...
m54099_200720_153206/4194505/ccs        272     U00096.3        567414  0       150=    *       0       0       GGACTGAGGGCAAAGGCCTCCCGGAAGTTCAGCCCGGT...
m54099_200720_153206/4194505/ccs        272     U00096.3        315863  0       150=    *       0       0       GGACTGAGGGCAAAGGCCTCCCGGAAGTTCAGCCCGGT...
...
m54099_200720_153206/4194627/ccs        0       U00096.3        274198  0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...
m54099_200720_153206/4194627/ccs        256     U00096.3        574834  0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...
m54099_200720_153206/4194627/ccs        256     U00096.3        688094  0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...
m54099_200720_153206/4194627/ccs        272     U00096.3        3130803 0       295=    *       0       0       CGGCCAACGAGCATGACCTCAATCAGCTGGGTAATCTG...
m54099_200720_153206/4194627/ccs        256     U00096.3        2101992 0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...
m54099_200720_153206/4194627/ccs        256     U00096.3        2289162 0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...
m54099_200720_153206/4194627/ccs        272     U00096.3        1396701 0       295=    *       0       0       CGGCCAACGAGCATGACCTCAATCAGCTGGGTAATCTG...
m54099_200720_153206/4194627/ccs        272     U00096.3        1300156 0       295=    *       0       0       CGGCCAACGAGCATGACCTCAATCAGCTGGGTAATCTG...
m54099_200720_153206/4194627/ccs        256     U00096.3        3365799 0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...
m54099_200720_153206/4194627/ccs        256     U00096.3        3652279 0       295=    *       0       0       CCCTTGTATCTGGCTTTCACGAAGCCGAACTGTCGCTT...

How do we decide the position? In the current implementation, the first subread of each molecule is taken (for details, see pacbio_data_processing.sm_analysis.map_molecules_with_highest_sim_ratio), because all the subreads are perfect. But notice that the positions (4th column) differ.