How do I analyze the data from LUTHOR HD 3’ mRNA-Seq libraries?

There are two ways/options to analyze your LUTHOR HD sequencing data:

1. You can use our Kangooroo platform, which offers data analysis pipeline for LUTHOR HD. Lexogen provides free of charge data analysis for customers with a valid voucher code. The voucher code is provided for each purchased LUTHOR HD library preparation kit. For more information on how to analyze your data with Kangooroo please visit our online FAQs.

2. You can analyze LUTHOR HD sequencing data by yourself by adapting our 3' mRNA-Seq data analysis pipeline available on our Github webpage (https://github.com/Lexogen-Tools/quantseqpool_analysis/blob/master/QuantSeqPoolAnalysis.sh).

A basic schematic of the steps involved in LUTHOR HD data analysis along with recommended tools can be found below.

For pipeline adjustment, please follow the instructions outlined below:

• Use our QuantSeq-Pool script available on our GitHub page (https://github.com/Lexogen-Tools/quantseqpool_analysis/blob/master/QuantSeqPoolAnalysis.sh), starting with UMI extraction from line 64 (i.e., skipping the first step ´´demultiplexing with idemux´´);

• Proceed with trimming (line 69) and alignment (line 74) steps as described on the GitHub page;

• Perform "gene tagging" of the alignments. You can do so by running:

  featureCounts -s 1  -t exon -g gene_id -a ${GTF} -o ${COUNT_FILE} -R BAM -M ${ALIGNED_BAM}

• Sort and index the tagged BAM, by following the code from line 82;

• For UMIs deduplication, use the modified command for positional and gene-based collapsing with UMI_tools (https://github.com/CGATOxford/UMI-tools): umi_tools dedup --umi-separator=":" --per-gene --gene-tag="XT" --stdin=${FEAUTRE_COUNTS_TAGGED_BAM} --stdout=${OUT_BAM}

• Finalize the data analysis with gene read counting (from line 92).

For differential gene expression analysis please use DESeq2.

Please contact support@lexogen.com for further information on how to analyze your LUTHOR 3' mRNA-Seq data.