Correction: the data in this article is produced with MNase rather than RNase I. They notice that although MNase is tolerated by fruitfly's ribosomes, MNase has a strong 3'A/T bias, which may yield a small amount of positional uncertainty. So most likely the sub-codon resolution is obsured by this.

Question: how could I use only annotated ORF in fasta? it seems all possible ORFs are used if I used findCDS.

Note: the pipeline I used below does not guarantee the reads in analysis are uniquely mapped to a certain genomic region. Cautions should be taken here.

Hi, I am trying to use riboSeqR on fly data, but what I got is not what I expected. I downloaded SRR942881.sra, and then preprocess it with cutadapt and trimmomatic. Then I mapped to rRNA, tRNA, pseudogene, ncRNA , miscRNA and miRNA to filter these reads out (flybase sequences dmel6.02). With only reads that can not mapped to rRNA and stuff, I mapped them to coding transcripts. Both are based on bowtie 1.1.2. But the tri-nucleotide periodicity is not very well as their original article (Joshua Dunn) states. Anything that I may have done wrong using riboSeqR

#download from flybase can not be trace, but it is from dmel 6.02
bowtie-build ../flybaseFasta/dmel-all-transposon-r6.02.fasta,../flybaseFasta/dmel-all-tRNA-r6.02.fasta,../flybaseFasta/dmel-all-pseudogene-r6.02.fasta,../flybaseFasta/dmel-all-miscRNA-r6.02.fasta,../flybaseFasta/dmel-all-miRNA-r6.02.fasta tRNAmimiPseuTransposon
bowtie-build ../flybaseFasta/dmel-all-transcript-r6.02.fasta transcript

# Now this dataset
wget ftp://ftp-trace.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR942/SRR942881/SRR942881.sra
fastq-dump SRR942881.sra

split -d -l 10257940 SRR942881.fastq SRR942881.fastq

for I in `seq -w 0 29`; do mv SRR942881.fastq${i} SRR942881.${i}.fastq; cutadapt -a CTGTAGGC -a ACTGTAGG -o SRR942881.trim${i}.fastq SRR942881.${i}.fastq & done

for I in `seq -w 0 29`; do java -jar ~/rd/software/Trimmomatic-0.33/trimmomatic-0.33.jar SE -phred33 SRR942881.trim${i}.fastq SRR942881.fine${i}.fastq ILLUMINACLIP:smallRNAv1.5.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20 2> SRR942881.fine${i}.err ; done

cat SRR942881.fine[0-9][0-9].fastq > SRR942881.fine.fastq

bowtie ~/rd2/data/flygenome/dmel_r6.02/bowtieIndex/tRNAmimiPseuTransposon SRR942881.fine.fastq -l 20 -p 12 --un SRR942881_bowtieNoRRNA.fastq > /dev/null

# reads processed: 76685044
# reads with at least one reported alignment: 44794396 (58.41%)
# reads that failed to align: 31890648 (41.59%)

bowtie ~/rd2/data/flygenome/dmel_r6.02/bowtieIndex/transcript SRR942881_bowtieNoRRNA.fastq -l 20 -p 12 --suppress 1,6,7,8 > SRR942881.bowtie.out

# reads processed: 31890648
# reads with at least one reported alignment: 22202258 (69.62%)
# reads that failed to align: 9688390 (30.38%)

# Now entering R

~/rd/software/R-3.2.3/bin/R

library("riboSeqR")

fastaCDS = findCDS(fastaFile="../fly-transcript-r6.02.fa" , startCodon = c("ATG"), stopCodon = c("TAG", "TAA", "TGA"))
riboDat = readRibodata("SRR942881.bowtie.out", replicates = c("wt3"))
fCs <- frameCounting(riboDat, fastaCDS , lengths = 27:35)
fS <- readingFrame(rC = fCs, lengths = 27:35)

plotFS(fS)

Thank you, Jason!

Sorry, wrong reply.