-- The previous approach (requiring > 5 copies among all reads) is breaking down in many samples (>1000) just from sequencing errors.
-- This breakdown is producing spurious clustered indels (lots of these!) around real common indels
-- The new approach requires >X% of reads in a sample to carry an indel of any type (no allele matching) to be including in the counting towards 5. This actually makes sense in that if you have enough data we expect most reads to have the indel, but the allele might be wrong because of alignment, etc. If you have very few reads, then the threshold is crossed with any indel containing read, and it's counted.
-- As far as I can tell this is the right thing to do in general. We'll make another call set in ESP and see how it works at scale.
-- Added integration tests to ensure that the system is behaving as I expect on the site I developed the code on from ESP
* restructured BQSR to report recalibrated tables.
* implemented empirical quality calculation to the BQSR stage (instead of on-the-fly recalibration)
* linked quality score quantization to the BQSR stage, outputting a quantization histogram
* included the arguments used in BQSR to the GATK Report
* included all three tables (RG, QUAL and COVARIATES) to the GATK Report with empirical qualities
On-the-fly recalibration with GATK Report
* loads all tables from the GATKReport using existing infrastructure (with minor updates)
* implemented initialiazation of the covariates using BQSR's argument list
* reduced memory usage significantly by loading only the empirical quality and estimated quality reported for each bit set key
* applied quality quantization to the base recalibration
* excluded low quality bases from on-the-fly recalibration for mismatches, insertions or deletions
-- This behavior, which isn't obviously valuable at all, continued to grab and rethrow exceptions in the HMS that, if run without NT, would show up as more meaningful errors. Now HMS simply checks whether the throwable it received on error was a RuntimeException. If so, it is stored and rethrow without wrapping later. If it isn't, only in this case is the exception wrapped in a ReviewedStingException.
-- Added a QC walker ErrorThrowingWalker that will throw a UserException, ReviewedStingException, and NullPointerException from map as specified on the command line
-- Added IT that ensures that all three types are thrown properly (i.e., you catch a NullPointerException when you ask for one to be thrown) with and without threading enabled.
-- I believe this will finally put to rest all of these annoying HMS captures.
-- Now you always get SNP and indel metrics with VariantEval!
-- Includes Number of SNPs, Number of singleton SNPs, Number of Indels, Number of singleton Indels, Percent of indel sites that are multi-allelic, SNP to indel ratio, Singleton SNP to indel ratio, Indel novelty rate, 1 to 2 bp indel ratio, 1 to 3 bp indel ratio, 2 to 3 bp indel ratio, 1 and 2 to 3 bp indel ratio, Frameshift percent, Insertion to deletion ratio, Insertion to deletion ratio for 1 bp events, Number of indels in protein-coding regions labeled as frameshift, Number of indels in protein-coding regions not labeled as frameshift, Het to hom ratio for SNPs, Het to hom ratio for indels, a Histogram of indel lengths, Number of large (>10 bp) deletions, Number of large (>10 bp) insertions, Ratio of large (>10 bp) insertions to deletions
-- Updated VE integration tests as appropriate
* fixed BadCigarFilter to filter out reads starting/ending in deletion and that have adjacent I/D events.
* added Unit tests for BadCigarFilter
* updated all exceptions in LocusIteratorByState to tell the user that he can instead run with -rf BadCigar
* added the BadCigar filter to ReduceReads and RealignTargetCreator (if your walker blows up with these malformed reads, you may want to add it too)
- Updated the documentation on the code
- Made the table.write() method private and updated necessary files.
- Added a constructor to GATKReport that takes GATKReportTables
- Optimized my code
Signed-off-by: Mauricio Carneiro <carneiro@broadinstitute.org>
This is important for quick turnaround in the analysis cycle of the new covariates. Also added a dummy unit test that doesn't really test anything (disabled), but helps in debugging.
Infrastructure:
* Added static interface to all different clipping algorithms of low quality tail clipping
* Added reverse direction pileup element event lookup (indels) to the PileupElement and LocusIteratorByState
* Complete refactor of the KeyManager. Much cleaner implementation that handles keys with no optional covariates (necessary for on-the-fly recalibration)
* EventType is now an independent enum with added capabilities. All functionality is now centralized.
BQSR and RecalibrateBases:
* On-the-fly recalibration is now generic and uses the same bit set structure as BQSR for a reduced memory footprint
* Refactored the object creation to take advantage of the compact key structure
* Replaced nested hash maps with single hash maps indexed by bitsets
* Eliminated low quality tails from the context covariate (using ReadClipper's write N's algorithm).
* Excluded contexts with N's from the output file.
* Fixed cycle covariate for discrete platforms (need to check flow cycle platforms now!)
* Redfined error for indels to look at the previous base in negative strand reads (using new PE functionality)
* Added the covariate ID (for optional covariates) to the output for disambiguation purposes
* Refactored CovariateKeySet -- eventType functionality is now handled by the EventType enum.
* Reduced memory usage of the BQSR script to 4
Tests:
* Refactored BQSRKeyManagerUnitTest to handle the new implementation of the key manager
* Added tests for keys without optional covariates
* Added tests for on-the-fly recalibration (but more tests are necessary)
Infrastructure:
* Generic BitSet implementation with any precision (up to long)
* Two's complement implementation of the bit set handles negative numbers (cycle covariate)
* Memoized implementation of the BitSet utils for better performance.
* All exponents are now calculated with bit shifts, fixing numerical precision issues with the double Math.pow.
* Replace log/sqrt with bitwise logic to get rid of numerical issues
BQSR:
* All covariates output BitSets and have the functionality to decode them back into Object values.
* Covariates are responsible for determining the size of the key they will use (number of bits).
* Generalized KeyManager implementation combines any arbitrary number of covariates into one bitset key with event type
* No more NestedHashMaps. Single key system now fits in one hash to reduce hash table objects overhead
Tests:
* Unit tests added to every method of BitSetUtils
* Unit tests added to the generalized key system infrastructure of BQSRv2 (KeyManager)
* Unit tests added to the cycle and context covariates (will add unit tests to all covariates)
-- TODO for ryan -- there are bugs in ActivityProfile code that I cannot fix right now :-(
-- UnitTesting framework for ActivityProfile -- needs to be expanded
-- Minor helper functions for ActiveRegion to help with unit tests
-- This report details which intervals are coming in and how many reads they contain
-- Added integration test to verify that the intervals aren't changing, before heading into the ART refactor
GATKReport format changes:
- All non-data header lines are preceeded with a single pound ( #:)
- Every report now has a report header containing the version number and number of tables
- Every table has two lines of table header: The first explains the size of the table and the data types of each column, the second contains the table name and description.
- This new format will allow reports in the future to be gatherable.
- Changed the header format to include an end-of-line string ":;"
Added features:
- Simplified GATK Reports:
The constructor for a simplified GATK Report. Simplified GATK report are designed for reports that do not need the advanced functionality of a full GATK Report.
A simple GATK Report consists of:
- A single table
- No primary key ( it is hidden )
Optional:
- Only untyped columns. As long as the data is an Object, it will be accepted.
- Default column values being empty strings.
Limitations:
- A simple GATK report cannot contain multiple tables.
- It cannot contain typed columns, which prevents arithmetic gathering.
- Added a constructor to generate simplified GATK reports.
- Added a method to easily add data to simple GATK reports.
- Upgraded the input parser take advantage of the new file format (v1).
- Added the GATKReportGatherer, more usability cmoing in next versionof GATK Report. Curently, it can only add rows from one table to another. Added private methods in GATKReport to combine Tables and Reports, It is very conservative and will only gather if the table columns, as well as everything else matches. At the column level, it uses the (redundant) row ids to add new rows. It will throw an exception if it is overwriting data.
- Made some GATKReport methods public, and added more setters and getters.
- Added method that compares formats of two GATKReports, and added an equals method to verify all data inside.
- The gsalib for R now supports reading GATKReport v1 files in addition to legacy formats (v0.*)
- Added a GATKReportDataType enum to give column a certain data type. This must be specified when making a gatherable report. This enum contains several methods including a reverse lookup map.
- Added a data type field in GATKColumn, when a type is not specified, the unknown type is used. Unknown types should not be gathered.
Test changes:
- Updated Unit Tests for GATK Report v1. Added a test for the gatherer. Left one test disabled while we transition from v0 to v1.
- Updated the MD5 hashes in integration tests throughout the GATK.
Other changes:
- Added the gatherer functions to CoverageByRG
- Also added the scatterCount parameter in the Interval Coverage script
- Dropped support for reading in legacy GATKReport formats ( v0.*)
- Updated VariantEvalWalker to work with GATK Report v1, added a format String to all applicable DataPoints.
- Rewrote the read file method for GATK report files.
- Optimized the equals methods within GATKReport. The protected functions should only be called by the GATKReport methods.
Signed-off-by: Mauricio Carneiro <carneiro@broadinstitute.org>
-- Refactored some duplicated code (FYI, code duplication = root of all evil) into shared functions
-- Added long-missing integrationtests
-- CHRIS/RYAN -- it would be very good to add an integration test covering external VCF files as I believe we rely on this functionality and it's not tested at all
GATKBAMIndex would allow an extremely confusing BufferUnderflowException to be
thrown when a BAM index file was truncated or corrupt. Now, a UserException is
thrown in this situation instructing the user to re-index the BAM.
Added a unit test for this case as well.
-- A cleaner table output (molten). For those interested in seeing how this can be done with GATKReports look here for a nice clean example
-- Integration tests
-- Minor improvements to GATKReportTable with methods to getPrimaryKeys
-- We weren't properly handling the case where a site had both a SNP and indel in both eval and comp. These would naturally pair off as SNP x SNP and INDEL x INDEL in eval, but we'd still invoke update2 with (null, SNP) and (null, INDEL) resulting most conspicously as incorrect false negatives in the validation report.
-- Updating misc. integrationtests, as the counting of comps (in particular for dbSNP) was inflated because of this effect.
Several of the unit tests for the new key authorization feature require
read access to the GATK master private key file. Since this file is only
readable by members of the group gsagit, this makes it hard for people
outside the group to run the test suite.
Now, we skip tests that require the master private key if the private
key exists (since not existing would be a true error) but is not readable
by the user running the test suite
Bamboo, of course, will always be able to run these tests.
-Running the GATK with the -et NO_ET or -et STDOUT options now
requires a key issued by us. Our reasons for doing this, and the
procedure for our users to request keys, are documented here:
http://www.broadinstitute.org/gsa/wiki/index.php/Phone_home
-A GATK user key is an email address plus a cryptographic signature
signed using our private key, all wrapped in a GZIP container.
User keys are validated using the public key we now distribute with
the GATK. Our private key is kept in a secure location.
-Keys are cryptographically secure in that valid keys definitely
came from us and keys cannot be fabricated, however keys are not
"copy-protected" in any way.
-Includes private, standalone utilities to create a new GATK user key
(GenerateGATKUserKey) and to create a new master public/private key
pair (GenerateKeyPair). Usage of these tools will be documented on
the internal wiki shortly.
-Comprehensive unit/integration tests, including tests to ensure the
continued integrity of the GATK master public/private key pair.
-Generation of new user keys and the new unit/integration tests both
require access to the GATK private key, which can only be read by
members of the group "gsagit".
-- Includes paired end status (T/F)
-- Includes count of reads used in calculation
-- Includes simple read type (2x76 for example)
-- Better handling of insert size, read length when there's no data, or the data isn't paired end by emitting NA not 0
-- ReadGroupProperties: Emits a GATKReport containing read group, sample, library, platform, center, median insert size and median read length for each read group in every BAM file.
-- Median tool that collects up to a given maximum number of elements and returns the median of the elements.
-- Unit and integration tests for everything.
-- Making name of TestProvider protected so subclasses and override name more easily
* Turns DNA sequences (for context covariates) into bit sets for maximum compression
* Allows variable context size representation guaranteeing uniqueness.
* Works with long precision, so it is limited to a context size of 31 bases (can be extended with BigNumber precision if necessary).
* Unit Tests added
-- DoC now by default ignores bases with reference Ns, so these are not included in the coverage calculations at any stage.
-- Added option --includeRefNSites that will include them in the calculation
-- Added integration tests that ensures the per base tables (and so all subsequent calculations) work with and without reference N bases included
-- Reorganized command line options, tagging advanced options with @Advanced
When aggregating raw BAM file spans into shards, the IntervalSharder tries to combine
file spans when it can. Unfortunately, the method that combines two BAM file
spans was seriously flawed, and would produce a truncated union if the file spans
overlapped in certain ways. This could cause entire regions of the BAM file containing
reads within the requested intervals to be dropped.
Modified GATKBAMFileSpan.union() to correct this problem, and added unit tests
to verify that the correct union is produced regardless of how the file spans
happen to overlap.
Thanks to Khalid, who did at least as much work on this bug as I did.
* added support to base before deletion in the pileup
* refactored covariates to operate on mismatches, insertions and deletions at the same time
* all code is in private so original BQSR is still working as usual in public
* outputs a molten CSV with mismatches, insertions and deletions, time to play!
* barely tested, passes my very simple tests... haven't tested edge cases.
premature push from my part. Roger is still working on the new format and we need to update the other tools to operate correctly with the new GATKReport.
This reverts commit aea0de314220810c2666055dc75f04f9010436ad.
- Added the GATKReportGatherer
- Added private methods in GATKReport to combine Tables and Reports
- It is very conservative and it will only gather if the table columns, match.
- At the column level it uses the (redundant) row ids to add new rows. It will throw an exception if it is overwriting data.
Added the gatherer functions to CoverageByRG
Also added the scatterCount parameter in the Interval Coverage script
Made some more GATKReport methods public
The UnitTest included shows that the merging methods work
Added a getter for the PrimaryKeyName
Fixed bugs that prevented the gatherer form working
Working GATKReportGatherer
Has only the functional to addLines
The input file parser assumes that the first column is the primary key
Signed-off-by: Mauricio Carneiro <carneiro@broadinstitute.org>
* new unit tests for the alignment shift properties of reduce reads
* moved unit tests from ReadUtils that were actually testing GATKSAMRecord, not any of the ReadUtils to it.
* cleaned up ReadUtilsUnitTest
Eric reported this bug due to the reduced reads failing with an index out of bounds on what we thought was a deletion, but turned out to be a read starting with insertion.
* Refactored PileupElement to distinguish clearly between deletions and read starting with insertion
* Modified ExtendedEventPileup to correctly distinguish elements with deletion when creating new pileups
* Refactored most of the lazyLoadNextAlignment() function of the LocusIteratorByState for clarity and to create clear separation between what is a pileup with a deletion and what's not one. Got rid of many useless if statements.
* Changed the way LocusIteratorByState creates extended event pileups to differentiate between insertions in the beginning of the read and deletions.
* Every deletion now has an offset (start of the event)
* Fixed bug when LocusITeratorByState found a read starting with insertion that happened to be a reduced read.
* Separated the definitions of deletion/insertion (in the beginning of the read) in all UG annotations (and the annotator engine).
* Pileup depth of coverage for a deleted base will now return the average coverage around the deletion.
* Indel ReadPositionRankSum test now uses the deletion true offset from the read, changed all appropriate md5's
* The extra pileup elements now properly read by the Indel mode of the UG made any subsequent call have a different random number and therefore all RankSum tests have slightly different values (in the 10^-3 range). Updated all appropriate md5s after extremely careful inspection -- Thanks Ryan!
phew!
-- Missing BAMs were appearing as StingExceptions
-- Missing VCFs were showing up as CommandLineErrors, but it's clearer for them to be CouldNotReadInputFile exceptions
-- Added integration tests to ensure missing BAMs, VCFs, and -L files are properly thrown as CouldNotReadInputFile exceptions
-- Added path to standard b37 BAM to BaseTest
-- Cleaned up code in SAMDataSource, removing my parallel loading code as this just didn't prove to be useful.
This is somewhat of an arbitrary decision, and is negotiable. I could see treating
GT:PL ./.:.
differently from
GT:PL .:0,3,6
but am not sure the worth of doing so.
* using the filter() instead of map() makes for a cleaner walker.
* renaming the unit tests to make more sense with the other unit and integration tests
* if the adaptor boundary is more than MAXIMUM_ADAPTOR_SIZE bases away from the read, then let's not clip anything and consider the fragment to be undetermined for this read pair.
* updated md5's accordingly
-- Call sets with indels > 50 bp in length are tagged as CNVs in the tag (following the 1000 Genomes convention) and were unconditionally checking whether the CNV is already known, by looking at the known cnvs file, which is optional. Fixed. Has the annoying side effect that indels > 50bp in size are not counted as indels, and so are substrated from both the novel and known counts for indels. C'est la vie
-- Added integration test to check for this case, using Mauricio's most recent VCF file for NA12878 which has many large indels. Using this more recent and representative file probably a good idea for more future tests in VE and other tools. File is NA12878.HiSeq.WGS.b37_decoy.indel.recalibrated.vcf in Validation_Data
Some tests in this class were intermittently not being executed due
to being randomly scheduled before tests whose results they depend on.
Now the serial dependencies are enforced to avoid problematic orderings.
* Knuth-shuffle is a simple, yet effective array permutator (hope this is good english).
* added a simple randomSubset that returns a random subset without repeats of any given array with the same probability for every permutation.
* added unit tests to both functions
* Modified cleanCigarShift to allow insertions in the beginning and end of the read
* Allowed cigars starting/ending in insertions in the systematic ReadClipper tests
* Updated all ReadClipper unit tests
* ReduceReads does not hard clip leading insertions by default anymore
* SlidingWindow adjusts start location if read starts with insertion
* SlidingWindow creates an empty element with insertions to the right
* Fixed all potential divide by zero with totalCount() (from BaseCounts)
* Updated all Integration tests
* Added new integration test for multiple interval reducing
These functions are methods of the read, and supplement getAlignmentStart() and getUnclippedStart() by calculating the unclipped start counting only soft clips.
* Removed from ReadUtils
* Added to GATKSAMRecord
* Changed name to getSoftStart() and getSoftEnd
* Updated third party code accordingly.
The algorithm wasn't accounting for the case where the read is the reverse strand and the insert size is negative.
* Fixed and rewrote for more clarity (with Ryan, Mark and Eric).
* Restructured the code to handle GATKSAMRecords only
* Cleaned up the other structures and functions around it to minimize clutter and potential for error.
* Added unit tests for all 4 cases of adaptor boundaries.
* New ClippingOp REVERT_SOFTCLIPPED_BASES turns soft clipped bases into matches.
* Added functionality to clipping op to revert all soft clip bases in a read into matches
* Added revertSoftClipBases function to the ReadClipper for public use
* Wrote systematic unit tests
Creating a single temporary directory per ant test run instead of a putting temp files across all runs in the same directory.
Updated various tests for above items and other small fixes.
fixed issue where a read starting with an insertion followed by a deletion would break, clipper can now safely clip the insertion and the deletion if that's the case.
note: test is turned off until contract changes to allow hanging insertions (left/right).
* fixed edge case when requested to hard clip beginning of a read that had hanging soft clipped bases on the left tail.
* fixed edge case when requested to hard clip end of a read that had hanging soft clipped bases on the right tail.
* fixed AlignmentStart of a clipped read that results in only hard clips and soft clips
note: added tests to all these beautiful cases...
* expanded the systematic cigar string space test framework Roger wrote to all tests
* moved utility functions into Utils and ReadUtils
* cleaned up unused classes
caught a bug in the hard clipper where it does not account for hard clipping softclipped bases in the resulting cigar string, if there is already a hard clipped base immediately after it.
* updated unit test for hardClipSoftClippedBases with corresponding test-case.
bug: When performing multiple hard clip operations in a read that has indels, if the N+1 hardclip requests to clip inside an indel that has been removed by one of the (1..N) previous hardclips, the hard clipper would go out of bounds.
fix: dynamically adjust the boundaries according to the new hardclipped read length. (this maintains the current contract that hardclipping will never return a read starting or ending in indels).
Eric Banks
Guillermo del Angel
Mark DePristo
Mauricio Carneiro
Menachem Fromer
Ryan Poplin
Roger Zurawicki
David Roazen
Khalid Shakir
Christopher Hartl
Matt Hanna
Laurent Francioli