####QUANTITATIVE DEVELOPMENT PIPELINE USED BY ECHINOBASE#### ##This document serves as a record of the quantitative development pipeline performed at Echinobase## ##The instructions should also allow other researchers to perform these same analyses using their own data## ##Please direct any queries to Saoirse Foley (saoirse@cmu.edu)## ##Last updated - 4th June 2020## ##PREREQUISITES## - Reference genome - Reference .gtf file - Paired end sequence files to quantify (or other format - tweak commands accordingly) - RSEM (https://github.com/deweylab/RSEM and http://deweylab.biostat.wisc.edu/rsem/README.html) - Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) ##STAGE 1 - BUILD RSEM REFERENCE## - Run the following: rsem-prepare-reference --gtf YourGTF.gtf --bowtie2 YourRefGenome.fna ProjectSuffix ##STAGE 2 - CALCULATE EXPRESSION## - The following command will yield FPKM and TPM data for your inputs: rsem-calculate-expression -p 8 --paired-end --bowtie2 --estimate-rspd --append-names --output-genome-bam file_1.fastq file_2.fastq refSpur5 ProjectSuffix ##STAGE 3 - BUILD BIGWIG TRACK FOR VISUALIZATION## - Dependencies: deeptools; samtools; - First, sort the BAM output (generated in stage 2) by running: samtools sort YourBam.genome.bam > YourSortedBam.bam -Then, index the BAM: samtools index YourSortedBam.bam - Optionally, convert your sorted BAM file to a smaller bigWig file: bamCoverage -b YourSortedBam.bam -o YourNewBigWigFile.bw