StringTie for Windows: Unofficial Community Build

This repository provides an unofficial Windows build of StringTie v3.0.3.

StringTie is a command-line program for transcript assembly and quantification from RNA-Seq alignments. The upstream project is primarily built for Unix-like environments. This repository vendors StringTie v3.0.3 as a patched source tree in stringtie-3.0.3.offline-patch/ and applies Windows/MSYS2-UCRT64 compatibility and reproducibility fixes.

These builds are not produced, endorsed, or supported by the upstream StringTie project. For StringTie itself, see the upstream repository:

https://github.com/gpertea/stringtie

Downloading StringTie for Windows

Prebuilt Windows binaries are available from the Releases page of this repository.

Download the latest release archive, for example:

stringtie-3.0.3-windows-ucrt64.zip

After extracting the archive, you should see:

stringtie-3.0.3-windows-ucrt64/
  stringtie.exe
  prepDE.exe
  libgcc_s_seh-1.dll
  libiconv-2.dll
  libintl-8.dll
  libstdc++-6.dll
  libwinpthread-1.dll
  libsystre-0.dll
  libtre-5.dll
  zlib1.dll
  README.md
  LICENSE.md
  THIRD_PARTY_NOTICES.txt
  LICENSES/

Keep the DLL files in the same folder as stringtie.exe.

How to Use

This Windows build uses the same command-line options as upstream StringTie. For detailed usage, options, and examples, refer to the upstream documentation:

https://ccb.jhu.edu/software/stringtie/

Example:

cd C:\Users\you\Downloads\stringtie-3.0.3-windows-ucrt64
.\stringtie.exe --version
.\stringtie.exe --help

Assemble transcripts from an indexed BAM file:

.\stringtie.exe -G annotation.gtf -o output.gtf alignments.bam

Generated text outputs from this Windows build are written with LF line endings.

prepDE.exe

prepDE.exe is a standalone Windows executable built from StringTie's upstream prepDE.py3 helper script. It generates gene-level and transcript-level count matrices from GTF files produced by stringtie -e. These CSV matrices are commonly used as input for downstream differential expression workflows such as DESeq2 or edgeR.

Typical workflow:

.\stringtie.exe -e -G annotation.gtf -o sample1\sample1.gtf sample1.bam
.\stringtie.exe -e -G annotation.gtf -o sample2\sample2.gtf sample2.bam

Create a sample list file, for example samples.txt:

sample1 sample1\sample1.gtf
sample2 sample2\sample2.gtf

Generate count matrices:

.\prepDE.exe -i samples.txt -g gene_count_matrix.csv -t transcript_count_matrix.csv

Use -l if the average read length is not the default value of 75:

.\prepDE.exe -i samples.txt -l 100

prepDE.exe can also scan a directory containing one subdirectory per sample:

.\prepDE.exe -i . -p "sample"

For all options:

.\prepDE.exe --help

Source Tree

The patched source tree is included in this repository:

stringtie-3.0.3.offline-patch/

The upstream StringTie README and license are kept inside that directory:

stringtie-3.0.3.offline-patch/README.md
stringtie-3.0.3.offline-patch/LICENSE

Patch notes and test notes are maintained in:

PATCHES.md
TEST_NOTES.md

Building from Source

You do not need to build StringTie yourself if you only want to use the released Windows binary. This section is for maintainers or users who want to recreate the build.

Install MSYS2 first. Open an MSYS2-UCRT64 shell and install the build tools and runtime dependencies:

pacman -S --needed \
  base-devel \
  mingw-w64-ucrt-x86_64-toolchain \
  mingw-w64-ucrt-x86_64-zlib \
  mingw-w64-ucrt-x86_64-libsystre

Build StringTie:

git clone https://github.com/win-ngs/stringtie-windows-build.git
cd stringtie-windows-build/stringtie-3.0.3.offline-patch
make release

The executable is created as:

stringtie-3.0.3.offline-patch/stringtie.exe

Validation Performed

This patched build was checked with MSYS2-UCRT64 using the upstream bundled test data. The final release build was confirmed to make all bundled tests #1 through #9 deterministic and identical to the expected GTF output.

The Windows-specific fixes are summarized in PATCHES.md. The most important runtime fixes are:

• use htslib's bundled random fallback instead of unavailable drand48()
• guard nascent-guide edge cases that could crash --nasc
• explicitly initialize GffObj bitfields that made guided/nascent output nondeterministic on UCRT64
• write generated text outputs with LF line endings on Windows