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Old 08-25-2016, 09:28 PM   #1
Len Trigg
Registered Vendor
 
Location: New Zealand

Join Date: Jun 2011
Posts: 29
Default RTG 3.7: Somatic calling improvements / targetted sequencing / variant comparison etc

Real Time Genomics are pleased to announce the availability of new releases of our full analysis suite, RTG Core, and our utility package, RTG Tools. This release includes new features and performance improvements. Some of the highlights of this release:

* Improvements to mapping speed when aligning targeted sequencing data. This feature makes use of a per-reference hash blacklist which is constructed once per reference genome and can yield significant speed improvement. In addition, several changes were made to reduce peak memory use during mapping.

* Variant callers now allow the optional inclusion of expected germline allele balance terms in the Bayesian model. In a genome-wide scale, this generally results in a reduction in false-positive calls, although sensitivity may be reduced for variants which do not follow allele balance expectations, such as mosaic de novo variants.

* Several improvements to the somatic caller. These include the ability to enable output of germline variants (due to the joint calling, accuracy of calling germline variants during somatic calling is typically higher than separately calling germline variants from the normal sample alone). The somatic caller now has the ability to explicitly model the expected somatic allelic fraction, for use in cases where the tumor heterogeneity is expected to be low. Additional options allow the output of records at sites exceeding user-specified thresholds for non-reference evidence. We have also included an AVR model specifically built for somatic calling which provides more accurate scoring than the regular germline AVR models.

* Several improvements to the variant comparison tools. vcfeval now includes the ability to evaluate matches across confident-region boundaries according to GA4GH recommended practise. vcfeval can be used to compare against "sample-free" VCFs such as ExAC/COSMIC/dbSNP, and the runtime has also been significantly improved. In addition, the rocplot command can now produce precision-sensitivity graphs, and can output SVG as a more publication-ready format.

If you haven't used RTG Core before (or maybe even if you have), we suggest you run the demo-family.sh script that runs through a short end-to-end demonstration of sex-aware and pedigree-aware family variant calling, including de novo variant detection and variant evaluation with vcfeval. (It also makes a nice demo of our comprehensive simulation tools.)

Commercial users of RTG Core may download the update from our website at http://realtimegenomics.com/products/rtg-core-downloads. Non-commercial users can download the update from our website at http://realtimegenomics.com/products...non-commercial or build from the source on github at https://github.com/RealTimeGenomics/rtg-core.

Users of RTG Tools, which is made freely available for non-commercial or commercial use alike, can download the new version from our website at http://realtimegenomics.com/products/rtg-tools or build from the source code on github at https://github.com/RealTimeGenomics/rtg-tools.

Note: RTG now requires Java 8, so for those using the "nojre" RTG download or who are building from source, make sure you have Java 8 installed.

Detailed changes are listed below by area. For more information on new features, see the RTG Operations Manual (which is now available in both PDF and HTML).

## Basic Formatting and Mapping

* format: Automatically installs reference genome configuration
information when a recognized reference genome is being formatted to
SDF. Also outputs a reminder for those cases where it looks like a
reference genome is being formatted but which is not one of the
recognized genomes.

* sdf2cg: New command to allow the export of Complete Genomics data
that has been formatted as SDF to Complete Genomics TSV read format.

* map/cgmap: TLEN was not being correctly computed in the presence of
soft clipping and back steps. This has now been corrected.

* map/cgmap: Several reductions in peak memory use during mapping.

* map: Significant speed improvement when mapping highly targeted
sequencing data, using the mechanism of a repetitive hash blacklist.
This is enabled via the new flag --reference-blacklist. A separate
tool 'hashdist' is used for this one-off blacklist construction.

* hashdist: New command that can be used to analyse the uniqueness of
k-mers contained within a reference sequence and to produce a
reference hash blacklist.

* calibrate: New flag --exclude-bed and --exclude-vcf can be used to
exclude sites of known genomic variation during the computation of
calibration data. It is not currently possible to specify this
information to the automatic calibration that is carried out during
mapping, this will be added in a future release.

### Variant Calling

* snp/family/population/somatic: These callers expect calling to be
carried out on alignments that have had calibration information
computed. They now requires the explicit use of the --no-calibration
flag in order to proceed anyway.

* snp/family/population/somatic: These commands now output a warning
if too many "excessive coverage" situations are encountered, as this
usually signifies that the user has incorrectly calibrated their
mappings or has failed to supply an appropriate coverage parameter
to the caller. In addition, these commands output a warning if it
appears that calibration has not been computed from correct regions
for targeted data.

* snp/family/population/somatic: New flag --min-base-quality which
allows explicit ignoring of base calls which do not meet the
specified minimum phred quality score. These bases will be treated
the same as an N and will not contribute to allele counts. The
default is to consider all bases.

* family/population/somatic: The semantics of --max-coverage has
changed from being the total coverage across all samples, to being
the average per-sample coverage. This flag is typically only used
when running without calibration, and this change makes the default
behaviour more scalable with varying numbers of samples.

* snp: An explicitly specified --ploidy flag now overrides the ploidy
obtained from reference genome configuration (if present).
Previously the ploidy specified in the reference genome would take
precedence.

* snp/family/population/somatic: Fixed an incorrect (and sometimes
non-deterministic) computation of the PUR FORMAT annotation. This
does not affect primary calling but could result in changes in AVR
score.

* snp/family/population/somatic: Updated the Bayesian model to include
a term for the expected allele balance. This is disabled by default,
and can be enabled with the new flag --enable-allelic-fraction. This
option gives improved precision for regular germline calling, but
sensitivity to mosaic variants or those within CNV regions may be
reduced.

* snp/somatic: The new flags --min-variant-allelic-depth and
--min-variant-allelic-fraction can be used to enable output at sites
where these thresholds are met, even if the caller would not
otherwise make a call. Note that this does not act as a filter to
prevent the caller from output at sites where these thresholds are
not met.

* somatic: New flag --include-germline which instructs the somatic
caller to also output variants which have been identified as
germline variants.

* somatic: New flag --enable-somatic-allelic-fraction which instructs
the Bayesian model to include a term for the expected somatic
allelic fraction in the calling. This flag is most appropriate when
tumor heterogeneity is low.

* somatic: A new pre-built AVR model is provided for somatic calling
which provides better scoring for somatic variants than the regular
AVR models. This new model, "illumina-somatic.avr" is selected by
default by the somatic caller.

### Variant Processing and Analysis

* vcfsubset/vcffilter: New flag --no-header which omits the output of
the VCF header.

* vcffilter: New option --keep-expr to allow filtering records based
on simple JavaScript expressions with natural VCF field access. For
example 'NA12878.DP > NA12892.DP' to select records from a trio
call-set where the depth of NA12878 is greater than that of her
mother. See the user manual for more information and examples.

* vcffilter: New option --javascript to allow advanced filtering and
other processing of the VCF file using powerful JavaScript
filters. These scripts can contain initial setup, per-record
actions, and end functions. See the user manual for more information
and examples.

* vcfeval: Specifying a sample name of ALT for either the baseline or
call sample name instructs vcfeval to match against all possible
non-ref diploid (or haploid if using --squash-ploidy) genotypes
possible from the declared ALTs. This permits matching against a VCF
that contains no sample column, for example to find hits against a
sample-free VCF such as ExAC or COSMIC.

* vcfeval: New flag --evaluation-regions, which adds support for
matching across high-confidence/false-positive regions such as those
supplied with GIAB or Illumina Platinum Genomes truth sets according
to GA4GH recommendations. In summary, only matches against baseline
variants within these regions count as true positives and only
non-matched call variants made within these regions count as false
positives.

* vcfeval: Now outputs additional true positive statistics for the
unweighted calls, so you can see the simple count of true positives
in call representation. When computing precision, this uses the
unweighted call count in the denominator, to reduce representation
bias in the precision.

* vcfeval: Significant speed increase (often 2x speed up for typical
WGS comparisons).

* vcfeval: New output mode 'roc-only' which skips the output of VCF
files and only produces the ROC data files and summary metrics. This
reduces run-time and the size of the output directories when doing
many runs.

* vcfeval: Command line score field specification permits INFO.<name>
form, for consistency with JavaScript expression notation, although
the old form of INFO=<name> is still supported.

* rocplot: Added the ability to plot precision-sensitivity graphs via
the new flag --precision-sensitivity. In the interactive GUI the
graph type can also be changed on the fly via a dropdown chooser.

* rocplot: Added the ability to output images in SVG format, both in
non-interactive mode via the new flag --svg, and when saving images
from the interactive GUI.

* rocplot: Improved the default labelling of curves by including the
score field if available.

* rocplot: The curve palette size has been increased in order to allow
easier differentiation when more than 8 curves are being displayed
at once.

* rocplot: (GUI) Fixed an annoying bug that could occur when trying to
edit the title of the plot or of the curves. Several other minor GUI
improvements have been made, such as the ability to use the
mouse-wheel to scroll large lists of curves.

### Other

* aview: Now defaults to showing base colors in the terminal. Use
--no-base-colors to disable this.

* aview: Better error handling for invalid SAM records.

* aview: New flag --print-soft-clipped-bases to display soft-clipped
bases.

* chrstats: New flag --output-pedigree that can be used to create a
default pedigree file based on the mappings of multiple samples,
using inferred sample sex where possible.

* many: In several cases where a flag could be specified multiple
times, it is now possible to supply a comma separated list of
values. These are indicated in the output of --help.

* many: Most utility commands which write VCF files now do so
asynchronously, often resulting in significant speed improvements.

* all: The distribution now includes an HTML version of the operations
manual in addition to the PDF version.

* all: The minimum Java requirement for RTG is now Java 8.
__________________
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Real Time Genomics
www.realtimegenomics.com
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Old 10-17-2016, 04:08 PM   #2
Len Trigg
Registered Vendor
 
Location: New Zealand

Join Date: Jun 2011
Posts: 29
Default

New stable releases are now available which include minor improvements and bug fixes.

The first of these is our full analysis suite, RTG Core 3.7.1. The changes in this version are listed below. Commercial users may download the update from our website at http://realtimegenomics.com/products/rtg-core-downloads. Non-commercial users can download the update from our website at http://realtimegenomics.com/products...non-commercial or build from the updated source code on github at https://github.com/RealTimeGenomics/rtg-core.

We have also produced updated builds of our utilities package, RTG Tools 3.7.1, which is made freely available for non-commercial or commercial use alike. More information and download links are available from our website at http://realtimegenomics.com/products/rtg-tools or build from the updated source on github at https://github.com/RealTimeGenomics/rtg-tools.


RTG Core 3.7.1 (2016-10-18)
---------------------------

This release primarily includes bugfixes and minor improvements:

* map/cgmap: Addresses a pathological case where a particular paired-end
read pair plus reference sequence could run for a disproportionately
long time in highly repetitive regions.

* vcfeval: Fixes a rare exception that could occur when a "too-hard"
region occurs right at the end of a reference sequence.

* rocplot: Fixes an exception that would occur when trying to plot the
result of evaluating a call set (containing variants) against a
baseline containing no variants.

* rocplot: (gui) When loading several files on startup, sometimes the
initial view would not be fully zoomed out. We now ensure that the
plot is zoomed out after the initial files are loaded.

* vcffilter: Fixes a regression in command line flag validation that
would cause a talkback exception if no input file was supplied rather
than presenting an appropriate message.

* vcfmerge: Fixes an exception that could occur when merging a mixture
of regular VCFs containing sample columns with sites-only VCFs.

* bgzip: Fixes an exception that could occur when decompressing from
stdin.

* Minor documentation fixes.
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Real Time Genomics
www.realtimegenomics.com
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