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Tags reads in a BAM file based on other BAM files.

Project Description

Readtagger

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Readtagger is a set of tools for describing the origin of reads in a sequenced genome. It can be used to verify and detect integration events of known, exogenous sequences (such as viruses) or endogenous sequences (such as transposons). In addition, there is the possibility of expanding this to de-novo detection without a priori knowledge of the inserted sequences).

Installation

pip install readtagger

Description

The readtagger module can be used as a tool for verifying transposable element (TE) insertions predicted by TEMP and TEPID, but it can also serve as the basis for detecting insertions using the findcluster command (see below). The readtagger module examines the origin of sequencing reads by aligning reads to multiple references and recording possible homologies as custom SAM tags. This works for single and paired-end reads, so that one obtains knowledge about a read that is aligned at a physical location in the genome as well as about its mate.

The readtagger command evaluates potential homologies, and (by default) discards homologies for alignments that do not improve the nucleotide usage, For example if the unclipped portion of an alignment has a homology with a transposable element, while we only care about the clipped, unaligned portion. Similarly, we discard homologies for aligned pairs that are well within the expected insert size. These precautions reduce noise in regions of the genome that have spurious homologies to transposable elements, as not all of these regions are masked by tools such as RepeatMasker.

To generate insertion calls, one can use the findcluster command.

findcluster operates in multiple phases, that are roughly:

  1. Divide the input file into approximately 100 KB bins. This allows parallelization over multiple CPU cores or machines. Splitting files may introduce artefacts when splitting in a cluster of tags, therefore we attempt to find regions that have no tagged alignments.
  2. For each region we determine clusters of tagged alignments. A new cluster is created once additional alignments are not overlapping the current cluster coordinates. This is a rough first-pass that often includes multiple actual insertions and/or separates clusters that belong together, but does summarize all tagged alignments. Additionally we generate clusters of soft-clipped reads (independent of transposable element homology).
  3. For each cluster, we ask whether there are any other clusters close by (the search area depends on the insert size distribution) that could potentially be merged because they provide evidence for the same insertion. All reachable clusters are tested by all-against-all alignments using edlib. Putative 5p and 3p sites are determined using a consensus alignment if split read evidence is available.
  4. We then ask whether the clusters are internally consistent. This means that alignments that point for example away from the putative insertion site are taken from the cluster and form a new, second cluster.
  5. We repeat steps 3 and 4 until the number of clusters at each pass stays constant.
  6. We assign soft-clipping clusters to transposable element clusters if any read in the soft clipping cluster is also member of a transposable element cluster. (This is important in a separate step that links transposable element insertion sites across samples).
  7. We then quantify the fragments that support a putative insertion (this includes alignments that did not reach the threshold for being tagged), the fragments that point against an insertion and the uninformative reads and we call genotypes and genotype likelihoods based on the evidence. Alignment evidence for an insertion and evidence against an insertion is tagged using separate tags, which allows for visual verification of the quantification. A VCF file describing the insertions, a BAM file containing relevant alignments and a fasta file of assembled sequences is written per 100 KB region.
  8. Merge individual files

Usage

To tag reads in file a.bam with file b.bam and output to path output.bam, type

readtagger --tag_file a.bam --annotate_with b.bam ----output_file output.bam

This will by default tag reads with the AD, AR, BD and BR tags, where the AD tag has detail mapping information for the current read, while the BD tag has the information for the mate. AR and BR contain the aligned reference (i.e chromosome). The first letter can be changed on a per-file basis by appending “:first_letter_read:first_letter_mate” to the file path. To change the above example into X for the read and Y for the mate, run:

readtagger --tag_file a.bam --annotate_with b.bam:X:Z ----output_file output.bam

To tag one bam file using multiple alignment files, run:

readtagger --tag_file a.bam --annotate_with b.bam:A:B c.bam:C:D ----output_file output.bam

Now reads that align in file b.bam will be tagged with AR, AD and BR, BD, while reads aligned in file c.bam are marked with CR, CD and DR, DD.

Advanced usage

To see the advanced options, type:

readtagger -h

Testing

If you modify readtagger, you can run all tests by running tox:

pip install tox
tox

History

0.4.10 (2018-03-30)

  • Include unmapped but tagged mates in veriefied tags
  • Update findcluster galaxy tool and fix softclip cluster ids
  • Use a unique ID as variant ID
  • Stop collection evidence once we reach 10000 reads
  • Speed up finding of soft clip clusters
  • Implement VCF output
  • Make loglevel configurable for findcluster script and add option to output log to file
  • Look for softclipped reads in a 15nt window and compare 5p clips by their end
  • Add script and tool to confirm/reject insertions
  • Refine the detection of TE clusters that are very close to each other
  • Verify that reads really support a specific insertion
  • Fix sorting to CRAM output
  • Move sorting of softclip clusters to merging phase
  • Skip finding softclipped clusters when skipping TE clusters
  • Annotate softclips as part of TEs
  • Embedd SoftClipClusterFinder in ClusterFinder
  • Fix softclipped positions when read contains deletions

0.4.9 (2018-01-23)

  • Fix deployment to PyPI

0.4.8 (2018-01-23)

  • Update test data output and allow :
  • Add edlib to requirements in setup.py
  • Add softclip finder test
  • Build on python-3.6
  • Make futures library conditional for python2
  • Drop temporary from requirements
  • Extend testcoverage
  • Drop external_bin from BamAlignmentWriter
  • Many small simplifications, bugfixes and enhaced tests
  • Improve reporting of 5p and 3p clips
  • Add some wigglespace for finding the most likely TSD position
  • Keep insertions associated with deletion intact
  • Add testcase for a cluster that should not be split
  • Fix if/else logic for genotypes
  • Skip “genomic sinks” with lots of TE evidence
  • Continue on RuntimeError
  • Improve splitting of input file
  • Need to fetch reads in the specified region if using external_bin=False
  • Don’t use external samtools when finding clusters
  • Identify decoy regions based on cluster density
  • Drop reraise_with_stack, doesn’t work on py3
  • Fix outdated min/max coordinates leading to dropped chunks
  • Re-raise any exceptions when processing chunks
  • Fix OrderedDict syntax for py2 compatibility
  • Improve logging when splitting input into chunks
  • Don’t remove read that isn’t present anymore
  • Fix return value when assembling too many reads
  • Fix limiting of region when using multiple threads
  • Report maximum MAPQ of read evidence for a cluster
  • Bump minimum MAPQ to 4 by default and make it configurable
  • Refactor cap3 assembly (so it can be exchanged more easily) and add limit to how many reads it will assemble
  • Fix and apply read_is_compatible to all read with BD tag
  • Generalize marking clusters as compatible or incompatible and apply at every cluster split or join
  • Estimate nref/nalt using overlap of start and end if start and end are more than 50nt apart
  • Skip clusters of reads that are inconsistent
  • Remove redundant parenthesis, fix typo
  • Allow non-proper pairs when counting evidence
  • Account for max. mate distance when joining cluster
  • Add new dependencies to conda recipe
  • Prevent joining clusters that we previously split explicitly
  • Don’t thread/cache joining of cluster
  • Use lru_cache for some cigar operations
  • Use cigar_to_max function consistently
  • Make use of new AlignmentHeader object (old method now very slow)
  • Use edlib align instead of Cap3Assembly
  • Fix evidence_against functionality
  • Output reads that count as non-support
  • Allow picking up location of reference_fasta via env var for quicker test execution
  • Fix 3p evidence bam, fix nref with 1 breakpoint
  • Update test-data
  • Assign left/right based on AD if AD and BD are set
  • Make counting more accurate, cleanup various Cluster counts and write out split reads found via evidence_for_five/three_p
  • Collect evidence for insertions
  • Fix a typo in get_breakpoint_sequence
  • Fix resolving consensus ties if tie contains N
  • Upgrade to pysam 0.14
  • Make split_ads a property since the splits can update
  • Fix typo in dumb_consensus help
  • Add IUPAC to nucleotides dict
  • Restructure non_evidence so that evidence for and against can be counted
  • Use reference_start instead of deprecated pos
  • Implement get_breakpoint_sequence as a method of TargetSiteDuplication
  • Add evidence_for function
  • Update planemo from 0.46.1 to 0.48.0
  • Refine the cluster merging logic
  • Fix the overlap calculation, in case the re-aligned contig ends up at a different position
  • Update test data output, genotype outputs with higher precision (sigh)

0.4.7 (2018-01-23)

  • Fix Exception that occurs when cluster doesn’t have an associated contig
  • Fix TE alignment logic when using pre-indexed transposon references
  • Control which reads extend a cluster during cluster refinement
  • Add a safeguard to avoid merging unrelated, far-away clusters

0.4.6 (2017-12-13)

  • Deploy to conda on py3 as well
  • Make sure cluster chunks are ordered
  • Avoid hangs due to expection in multiprocessing tasks

0.4.2 (2017-12-13)

  • Fix passing of region specification to pileup engine
  • Point out typical useage of –reference_fasta and –reference_index
  • Fix cheetah bwa index variable for findcluster galaxy tool

0.4.1 (2017-11-20)

  • Add matplotlib and pandas to dependencies
  • Add a script that can plot coverage as an area plot between two bam files
  • Update dependencies
  • If either three_p or five_p of a tsd is unknown assign the available use the available side to test of a read belongs to the left or right side of an insertion
  • Fix crash for unaligned(?) reads
  • Change deprecacted alen, pos and mpos to current replacements
  • Tune clusterfinding for misaligned long reads

0.4.0 (2017-11-09)

  • Fixes for CRAM input and output
  • Adjust chunk-size in readtagger based on readlength (for pacbio/nanopore reads)
  • Cleanup temporary bwa indexes
  • Dependency updates

0.3.25 (2017-06-21)

  • Refine cluster coordinates using an Assembly strategy
  • Fix GFF sorting on python 3
  • Improve BWA alignment settings (default to intractg plus -Y) and add align_contigs method to SimpleAligner
  • Add pysamtools_view command
  • Improve cluster-splitting
  • Add multiprocessing-logging recipe
  • Only output BWA stderr if the exit code is not zero
  • Add a function to sort gff files
  • Close open file descriptors
  • Make imprecise insertion sites more realistic
  • Fix read_index property
  • Adapt readtagger to higher coverage datasets
  • Fix readtagger crash when not producing discard tag file.
  • Add number of mates for left and right support to GFF
  • Split clusters that start with reverse reads conatining only BD tags

0.3.24 (2017-05-11)

  • Split cluster if there are multiple polarity switches between Forward and Reverse orientation
  • Manipulate copy of cigarlist to avoid numpy issue

0.3.23 (2017-05-09)

  • Expose reference fasta option in bam_readtagger.xml

0.3.22 (2017-05-09)

  • Move readtagger CLI form argparse to click
  • Index bamfile if neccesary
  • Replace multipocessing pool with ProcessPoolExecutor
  • Set the matesequence while tagging reads
  • Fix false positives in readtagger module
  • Do cap3 assembly in shared memory if passing –shm_dir or if SHM_DIR environment variable is defined
  • Parallelize findlcluster by splitting input bam
  • Add check_call.py script for rapidly verifying IGV screenshots

0.3.21 (2017-04-27)

  • Fix crash when determining reference name

0.3.20 (2017-04-27)

  • Guess the best TE match and write it into GFF Parent
  • Fix case where input files are already sorted
  • Remove blast from requirements

0.3.19 (2017-04-27)

  • Skip creating tempdirs in current working directory
  • Remove blast-specific files
  • Switch to using BWA for annotating detected insertions
  • Add more logging and default to not changing sort order unless specifically demanded
  • Do dovetailing on coordinate-sorted file

0.3.18 (2017-04-25)

  • Fix small outputs due to switching of -t and -a options

0.3.17 (2017-04-25)

  • Fix file seeking
  • Update dependencies

0.3.16 (2017-04-23)

  • Parallelize readtagger

0.3.15 (2017-04-20)

  • Do not count reads as support if both AD and BD tag contribute to an insertion
  • Remove sambamba support

0.3.14 (2017-04-19)

  • Perform readtagging on readname sorted files.
  • Catch possible errors
  • Add BWA alignment module to replace Blast

0.3.13 (2017-04-05)

  • Add possibility to output cluster contigs as fasta

0.3.12 (2017-03-31)

  • Fix and accelerate the calculation of nref (=non support evidence)
  • Update priors and genotype frequrencies to a more realistic model

0.3.11 (2017-03-28)

  • Add a testcase for genotyping module
  • Stream over full alignment file instead of fetching regions, pysam.AlignmentFile.fetch is too slow

0.3.10 (2017-03-26)

  • Revert local conda dependency resolution
  • Fix readtagger.add_mate to work also if one mate is unmapped

0.3.9 (2017-03-26)

  • Add a genotyping module
  • Keep tags for alternative alignments if mates are not in a proper pair

0.3.4 (2017-03-02)

  • Speed up assembly steps using multithreading
  • Implement a cache for the Cluster.can_join method

0.3.3 (2017-03-02)

  • Fix a crash when writing GFF for a cluster of hardclipped reads
  • Change confusing variable names and copypasted docstring

0.3.2 (2017-03-02)

  • Fix another crash when tuple starts with 1,2,7 or 8

0.3.1 (2017-03-02)

  • Fix a crash when a mismatch is the last item in a cigartuple

0.3.0 (2017-03-02)

  • Add a galaxy tool for the findcluster script
  • Add new script that finds clusters of reads and outputs GFF or BAM files with these clusters.
  • Implement writing clusters as GFF files
  • Implement writing out reads with cluster number annotated in CD tag.
  • Implement merging of clusters based on whether reads contribute to common contigs
  • Use cached-property where it makes sense
  • Add module to find, join and annotate clusters of reads
  • Represent cigartuple as namedtuple
  • Add a Roadmap file
  • Add more logic for finding ends of insertions and
  • Manipulate cluster of reads to find TSDs
  • Add module for cap3 assembly and manipulation of assembled reads
  • Fix conda recipe script entrypoints

0.2.0 (2017-02-21)

  • Reformat help text in galaxy wrappers
  • Add add_matesequence script to add the sequence of the mate of the current read as a tag
  • Add option to discard alternative tag if read is a proper pair
  • Stitch cigars that are separated by I or D events
  • Add a tag tuple that knows how to format itself
  • Update README.rst example with current default tag prefix
  • Test with and without discarding verified reads
  • Symlink test-files that are shared with the galaxy test, add testcase for allow_dovetailing script
  • Fix HISTORY.rst formatting

0.1.13(2017-02-17)

  • Add instructions for development
  • Install planemo in deployment step

0.1.12(2017-02-17)

  • Test deployment again

0.1.11 (2017-02-17)

  • Test deployment

0.1.10 (2017-02-17)

  • Fix toolshed deployment

0.1.9 (2017-02-17)

  • Add automated deployment to Galaxy Toolshed
  • Add instructions for development and release process

0.1.8 (2017-02-17)

  • Minor release to test release process

0.1.7 (2017-02-17)

  • Extend testing with coverage testing
  • Automate deployment to pypi and conda
  • Register project with pyup.io

0.1.6 (2017-02-16)

  • Rename to readtagger
  • Fix bug with stdin closing file descriptor too early, leading to corrupt BAM files
  • Extend testing

0.1.5 (2017-02-12)

  • Add option (-wd) to write suboptimal tag into separate BAM file
  • Add option (-wv) to write verified tags into separate BAM file
  • Performance improvments by letting sambamba handle BAM reading and writing. Also elimininate regualr expression to parse cigarstring

0.1.4 (2017-02-10)

  • Add option (-k) to keep alternative tags if they do not explain the softclipped read any better. Default is to discard them.

0.1.3.2 (2017-02-08)

  • Fix dovetailing script

0.1.3 (2017-02-07)

  • Add option to allow dovetailing in alignment files when tagging reads
  • Add separate entrypoint for standalone script

0.1.2 (2017-02-05)

  • Add conda recipe
  • Python3 string fix

0.1.0 (2017-02-05)

  • Initial version

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