Basecall Fast5 reads using flip-flop basecalling.

• Flip-flop basecalling for the MinION platform
  • R9.4.1 (Native or PCR libraries)
  • R10C (PCR libraries only)
• Basecalling of 5mC in CpG context for R9.4.1, PromethION platform

Flappie has been tested on Ubuntu 16.04.5 LTS. Other systems may be compatible.

Flappie models and other large resources are stored using git lfs and this extension must be installed to successfully clone the repository.

git clone https://github.com/nanoporetech/flappie
cd flappie
make flappie

Flappie has the following dependences

• Cmake for building
• CUnit library for unit testing
• HDF5 library
• OpenBLAS library for linear algebra

On Debian based systems, the following packages are sufficient (tested Ubuntu 14.04 and 16.04)

• Running
  • libcunit1
  • libhdf5
  • libopenblas-base
• Building
  • cmake
  • libcunit1-dev
  • libhdf5-dev
  • libopenblas-dev

#  ! It is highly recommended that OpenBLAS is run in single threaded mode
export OPENBLAS_NUM_THREADS=1
#  List available models
flappie --model help
#  Basecall reads directory
flappie reads/ > basecalls.fq
#  Basecall using a different model
flappie --model r941_5mC reads/ > basecalls.fq
#  Output to SAM (not compatible with modification calls)
flappie --format sam reads/ > basecalls.sam
#  Output to BAM (not compatible with modification calls)
flappie --format sam reads | samtools view -Sb - > basecalls.bam
#  Dump trace data
flappie --trace trace.hdf5 reads > basecalls.fq
#  Basecall in parallel
find reads -name \*.fast5 | parallel -P $(nproc) -X flappie > basecalls.fq
#  Dump trace in parallel.  One trace per parallel process.
find reads -name \*.fast5 | parallel -P $(nproc) -X flappie --trace trace_{%}.hdf5 {} > basecalls.fq

(c) 2018 Oxford Nanopore Technologies Ltd.

Flappie is distributed under the terms of the Oxford Nanopore Technologies, Ltd. Public License, v. 1.0. If a copy of the License was not distributed with this file, You can obtain one at http://nanoporetech.com

The vectorised math functions used by Flappie src/sse_mathfun.h are from http://gruntthepeon.free.fr/ssemath/ and the original version of this file is under the 'zlib' licence. See the top of src/sse_mathfun.h for details.

If you encounter compilation failures of the following form, the repository was cloned without git lfs and the model files are missing.

/home/ubuntu/mounted/extensionBonusFlappie/flappie/src/models/flipflop_r941native.h:1:1: error: unknown type name ‘version’
version https://git-lfs.github.com/spec/v1
^~~~~~~
/home/ubuntu/mounted/extensionBonusFlappie/flappie/src/models/flipflop_r941native.h:1:14: error: expected ‘=’, ‘,’, ‘;’, ‘asm’ or ‘__attribute__’ before ‘:’ token
version https://git-lfs.github.com/spec/v1
              ^
/home/ubuntu/mounted/extensionBonusFlappie/flappie/src/models/flipflop_r941native.h:2:12: error: invalid suffix "fa49fa0ea6c67806f69fd6ba42a7dd935390b86f98a615ee03e167d22583" on integer constant
oid sha256:3979fa49fa0ea6c67806f69fd6ba42a7dd935390b86f98a615ee03e167d22583
            ^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
In file included from /home/ubuntu/mounted/extensionBonusFlappie/flappie/src/networks.c:4:0:
/home/ubuntu/mounted/extensionBonusFlappie/flappie/src/models/flipflop_r10Cpcr.h:2:12: error: invalid suffix "b2fe5fd1c3d9646e7a6ea76e646beb50ad6a5fe17e5da0c76c13bd907cb4" on floating constant
oid sha256:83e2b2fe5fd1c3d9646e7a6ea76e646beb50ad6a5fe17e5da0c76c13bd907cb4
            ^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Extremely high system load can arise when Flappie is run using parallel and OpenBLAS is used in multi-threaded mode. Running in this manner is harmful to overall throughput and it is recommended that OpenBLAS is used in single-threaded manner, which can be enable by setting the OPENBLAS_NUM_THREADS environmental variable to 1 (see top of Usage for an example of how to do this).

From git lfs, the installation instructions for Debian based systems, like Ubuntu, are:

curl -s https://packagecloud.io/install/repositories/github/git-lfs/script.deb.sh | sudo bash
sudo apt-get install git-lfs
git lfs install

Flappie currently only calls 5mC methylation in CpG contexts. Calling other modifications, or 5mC in other contexts, is not currently supported.

Methylated calls are currently represented as a 'Z' base in the output -- this is likely to break down-stream tools and may not be final format in which this modification information is represented. Outputting methylation from Flappie is enabled for early adopters to think about how this information may be used; please do not rely on this particular representation of modifications as it may change in the future. Particularly, the SAM output when modification calling is enabled does not conform to the published specification for that for format.

See nanoporetech#11 and samtools/hts-specs#362 for more details of the issues involved.

The models contained contained in Flappie are trained using data from the MinION platform. Use on other platforms is not supported, although they may generalise to reads from the GridION platform due to the similarity of the hardware.

The quality currently produced for FASTQ and SAM output are derived directly from the probabilistic model output by the Flappie model and have not been calibrated.

The trace information is output as a block x state matrix, where the states are the flip (uppercase) and flop (lowercase) bases in the order ACGTacgt or ACGTZacgtz for methylated calls. The probabilities for each state are normalised into the range 0..255 and then represented by an unsigned 8bit integer. Due to rounding, the sum of encoded probabilities for each block may not equal 255.