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

Our LUTHOR HD V2 kit includes a voucher code for 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.

NOTE: Our LUTHOR HD pipeline utilizes paired-end data only! Please, upload both Reads (1 and 2) FASTQ files for your data analysis!

If you prefer to analyze your LUTHOR HD data on your own, as reference you can find below the general workflow and pipeline adjustments to successfully analysis your data!

Key steps of the general workflow to use to analyze LUTHOR HD data:

As reference, you can use our 3' mRNA-Seq data analysis pipeline available on our Github webpage (https://github.com/Lexogen-Tools/quantseqpool_analysis/blob/master/QuantSeqPoolAnalysis.sh) with the following adjustments:

• Start with UMI extraction from line 64 (i.e., skipping the first step ´´demultiplexing with idemux´´). Please apply the following changes:

o   "extracting N10 UMIs from read 2" to "extracting N12 UMIs from read 2"

o   --bc-pattern2 NNNNNNNNNN to --bc-pattern2 NNNNNNNNNNNN

• 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.