Alignment Commands:

1. ILMN (Illumina)

(1) STAR
star --runThreadN 1 --genomeDir hg19_gencodeV12_50 --readFilesIn input.R2.fastq.gz input.R2.fastq.gz --outFileNamePrefix ABRF-ILMN-RIBO-A-1_D1DNBACXX_CTGAAGCTATAGAGGC_L001_R1_001. --outStd SAM --outSAMunmapped Within --outFilterMultimapNmax 1 --chimSegmentMin 20 --chimScoreMin 1

(2) ELAND
ELAND_standalone.pl-if $input.R1.fastq.gz -if input.R2.fastq.gz -ref hg19/ --bam -it FASTQ -od . -op output -rt -l output.log

2. PGM and PRO (Life Technology)

(1) TMAP
tmap map all -f hg19.fa -r <(zcat input.fastq.gz) -i fastq -Y -a 0-o 1 -g 0 -n 5 -s output.bam stage1 map1 map2 map3

(2) STAR
star-long --genomeDir hg19_gencodeV12_100 --genomeLoad LoadAndKeep --readFilesIn input.fastq.gz --readFilesCommand zcat --outFileNamePrefix output. --outStd SAM --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMismatchNmax 8 --outFilterMismatchNoverLmax 0.3 --seedSearchLmax 30 --seedSearchStartLmax 30 --seedPerReadNmax 100000 --seedPerWindowNmax 100 --alignTranscriptsPerReadNmax 100000 --alignTranscriptsPerWindowNmax 10000 --chimSegmentMin 20 --chimScoreMin 1

3. PAC (Pacific Biosciences)

(1) GMAP
gmap -D gmap_db/ -d hg19 -t 24 -f samse -n 0 input.fastq.gz

(2) STAR
star-long --genomeDir hg19_gencodeV12_100 --genomeLoad LoadAndKeep --readFilesIn input.fastq.gz --readFilesCommand zcat --outFileNamePrefix output. --outStd SAM --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMismatchNmax 100 --outFilterMismatchNoverLmax 0.3 --seedSearchLmax 30 --seedSearchStartLmax 30 --seedPerReadNmax 100000 --seedPerWindowNmax 100 --alignTranscriptsPerReadNmax 100000 --alignTranscriptsPerWindowNmax 10000 --chimSegmentMin 20 --chimScoreMin 1

4. 454 (Roche)

(1) GS Reference Mapper (GUI with "cDNA" settings)

(2) STAR
star-long --genomeDir hg19_gencodeV12_100 --genomeLoad LoadAndKeep --readFilesIn input.fastq.gz --readFilesCommand zcat --outFileNamePrefix output. --outStd SAM --outSAMunmapped Within --outFilterMultimapNmax 1 --outFilterMismatchNmax 50 --outFilterMismatchNoverLmax 0.3 --seedSearchLmax 30 --seedSearchStartLmax 30 --seedPerReadNmax 100000 --seedPerWindowNmax 100 --alignTranscriptsPerReadNmax 100000 --alignTranscriptsPerWindowNmax 10000 --chimSegmentMin 20 --chimScoreMin 1

R-Scripts for Calculating Differentially Expressed Genes:

R Libraries needed for the R scripts:
library(sva)
library(limma)
library(edgeR)
library(raster)
library(doMC)
library(ggplot2)

  1. PolyA
  2. RIBO
  3. 454
  4. PGM
  5. PRO

ABRF functions Rscript - All R functions used for ABRF and MCC analysis

CV Rscript- Rscript used for CV calculation. Shuffling methods could be found here

RScript for the DEG analysis using comparable exome coverage between RIBO and POLYA - for files used, click here

Venn diagram of DEGs shared among platforms