Read Quality Recalibrator
-------------------------
The tools in this package recalibrate quality scores
assigned to nucleic acids in an aligned BAM file by
analyzing the covariation between machine reported
quality scores and:
1) the position within the read, and
2) the preceding nucleotide (sequencing chemistry effect).
The aligned reads have their dbSNP sites masked out, and
the mismatched bases are used as a metric for the true error
rate of the system. The error rate at different dinucleotides
and positions in the read is then fed into a logistic regression
system which outputs a correction factor for each of those
combinations which are then use to output a recalibrated BAM
file.
Software Dependencies
---------------------
The recalibrator currently depends on the following
applications. Please install these before proceeding.
- Java Runtime Environment 1.6.0_12 or later
- Python 2.4.2 or later
- R 2.6.0 or later
- samtools 0.1.4 or later.
Please update the file paths at the top of
RecalQual.py to point to the local installations of the
software listed above.
Running
-------
Before running the tool, please update the file paths
listed at the top of RecalQual.py to point to the
local installations of the tools listed above.
The recalibrator has two modes: recalibration and
evaluation, which can be run separately or jointly.
By default, the recalibrator will recalibrate only.
To calibrate a given bam file, run the following
command:
python RecalQual.py <source bam> <recalibrated bam>
After recalibration, performing evaluation will walk
through the source BAM file again, remeasuring the
differences between empirical vs. reported quality.
The results of evaluation are comma-delimited text
files (.csv) and graphs (.png) which can be found
in the output directory (see below) for the given run.
A successful recalibration should produce a plot of
empirical vs. observed qualities that shows little
difference between the two values.
To both recalibrate and evaluate, execute:
python RecalQual.py --recalibrate --evaluate <source bam> <recalibrated bam>
To (only) evaluate a given bam file after calibrating:
python RecalQual.py --evaluate <source bam> <recalibrated bam>
Output
------
The recalibration process keeps many incremental
files around for future analysis. By default, all
of these files will be grouped into the directory
'output.<source bam>/'. By default, this directory
will be created within the working directory. This
location can be changed by editing the output_root
variable at the top of RecalQual.py.
It is safe to delete this supplemental output
directory at any time.
Known Issues
------------
- The recalibrator places severe memory demands on
files with large numbers of read groups (> 1000).
- If running in 'evaluation' mode (see the 'Running'
section above), X11 is required to generate the
graphs. If running on a machine via ssh, be certain
to enable X tunnelling.
Support
-------
For support, please email gsadevelopers@broad.mit.edu.
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