How can I analyze my QuantSeq-Pool data?

Our QuantSeq-Pool kit comes with a voucher code to access free data analysis on our web-based platform Kangooroo.

For more information, please visit our website Lexogen - Kangooroo and our online Kangooroo Data AnalysisFAQs!

If additional voucher codes are needed, please contact us at sales@lexogen.com.

ATTENTION: Kangooroo only accepts demultiplexed FASTQ files. QuantSeq-Pool libraries contain an inline i1 barcode that cannot be demultiplexed by standard Illumina tools (e.g., bcl2fastq or BCL-convert), which only process external i7/i5 indices. Before uploading data to Kangooroo, please demultiplex the UDI indices (if used) and the i1 barcode. For i1 demultiplexing, we recommend using Idemux.

NOTE: Our QuantSeq-Pool pipeline utilizes paired-end data. Please, upload both Read 1 and Read 2 FASTQ files.

If you prefer to analyze your QuantSeq-Pool data on your own, as reference you can find below the general workflow with key steps to successfully analysis your QuantSeq data!

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

Alternatively, Lexogen also offers the 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.