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 see https://faqs.lexogen.com/faq/kangooroo-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.