The program Flexbar preprocesses high-throughput sequencing data efficiently. It demultiplexes barcoded runs and removes adapter sequences. Moreover, trimming and filtering features are provided. Flexbar increases read mapping rates and improves genome as well as transcriptome assemblies. Unique molecular identifiers can be extracted in a flexible way. The program supports sequencing data in fasta and fastq format, e.g. from the Illumina platform.
Refer to the manual or contact Johannes Roehr for support with this application.
Johannes T. Roehr, Christoph Dieterich, Knut Reinert: Flexbar 3.0 – SIMD and multicore parallelization. Bioinformatics 2017.
See article on PubMed
Matthias Dodt, Johannes T. Roehr, Rina Ahmed, Christoph Dieterich: Flexbar – flexible barcode and adapter processing for next-generation sequencing platforms. Biology 2012.
See article on PubMed
Flexbar source code as well as binaries for Linux and Mac OS can be downloaded on the release page. Please follow instructions for building or setup of binaries below. Additionally, Flexbar is available via package manager on Debian systems. Versions before 2.4 can be found on the old page.
Make sure that cmake is available, as well as development and runtime files of the TBB library 4.0 or later (Intel Threading Building Blocks). For example on Debian systems, install the packages libtbb-dev and libtbb2. Furthermore, the SeqAn library and a compiler that supports C++14 is required:
Decompress both files:
tar xzf flexbar-3.2.0.tar.gz
tar xJf seqan-library-2.2.0.tar.xz
Move SeqAn include folder to Flexbar:
mv seqan-library-2.2.0/include flexbar-3.2.0
Use these commands for building:
cd flexbar-3.2.0
cmake .
make
Flexbar version 2.7 requires SeqAn 2.1.1 instead. Releases prior to 2.7 use the SeqAn 1.4.2 library.
For execution of provided Flexbar binaries, the corresponding TBB library has to be available. Downloads contain the library file for runtime. Follow the platform specific instructions below.
Adjust lib search path to include the absolute path of the Flexbar directory containing the lib file libtbb.so.2 for the current terminal session, or permanently in shell startup scripts:
export LD_LIBRARY_PATH=/path/FlexbarDir:$LD_LIBRARY_PATH
It applies the same as for Linux. Make the file libtbb.dylib available by setting the lib search path:
export DYLD_LIBRARY_PATH=/path/FlexbarDir:$DYLD_LIBRARY_PATH
Flexbar needs at least one file with sequencing reads in fasta or fastq format as input. Additionally, the target name and further options can be specified. For read separation based on barcodes and for adapter removal, a file in fasta format with barcode or adapter sequences should be provided.
flexbar -r reads [-b barcodes] [-a adapters] [options]
Refer to the help screen flexbar -h or manual for more information. Although default parameters of Flexbar are optimized to deliver good results in many scenarios, the adjustment of parameters might improve results, e.g. --adapter-min-overlap. To run tests, make sure flexbar is reachable via the path variable and run flexbar_test.sh within the test folder.
In this example, reads that are barcoded on left side are demultiplexed by specifying a file with barcodes in fasta format. After separation of reads, given adapters are removed from the right side if they do not align before read start. The left side of reads is kept if long enough. Remaining reads are written to the file target.fastq in same format as the input.
flexbar -r reads.fq -t target -b brc.fa -be LTAIL -a adp.fa
The second example shows how to trim compressed reads based on their quality scores in illumina version 1.8 format. Afterwards, provided adapters are removed in right trim-end mode, only if the overlap of adapter and read has at least length 5 with at most 20% errors.
flexbar -r reads.fq.gz -q TAIL -qf i1.8 -a adp.fa -ao 5 -at 0.2
For further examples visit the manual page.