How do I analyze my QuantSeq-Pool data?

Lexogen offers a specific QuantSeq-Pool data analysis pipeline, which can be found on our GitHub page.

The script QuantSeqPoolAnalysis.sh uses the following software tools and scripts in its routine. These will need to be installed prior to use:

    - idemux (https://github.com/Lexogen-Tools/idemux)
    - umi_tools (https://github.com/CGATOxford/UMI-tools)
    - cutadapt (https://cutadapt.readthedocs.io/en/stable/)
    - STAR aligner (https://github.com/alexdobin/STAR)
    - samtools (https://www.htslib.org/)
    - featureCounts (http://subread.sourceforge.net/)
    - GNU awk (https://www.gnu.org/software/gawk/)

Following demultiplexing which requires the use of Lexogen’s idemux tool (available in C++ or Python versions), in principle, all further steps for trimming, mapping, UMI processing, and counting can be integrated into existing standard data analysis pipelines. For further questions about the pipeline, please contact support@lexogen.com.

Important to note when analyzing QuantSeq-Pool libraries are the following:

• QuantSeq Pool libraries include the Read 1 linker sequence in the 5' part of the second strand synthesis primer, hence NGS reads are generated towards the poly(A) tail and reflect the sequence of the corresponding mRNA.

• The 10 nt UMI sequence and subsequent 12 nt sample barcodes must be read out in read 2, therefore paired-end reads are required as input for QuantSeq-Pool data analysis.

Key steps of the general workflow to use to analyze QuantSeq-Pool data: