How to format input data for Picard CollectRnaSeqMetrics

Question

[Lina F]

Hi all,

I am trying to run Picard's CollectRnaSeqMetrics tool and noticed that my output contains a lot of zero values. I only get values for the following fields:

PF_BASES -------------> 553934269
PF_ALIGNED_BASES -----> 552090508
RIBOSOMAL_BASES ------> 6920715
INTERGENIC_BASES -----> 545188347
PCT_RIBOSOMAL_BASES --> 0.012535
PCT_INTERGENIC_BASES -> 0.987498

All the other values are zero.

I am also a little bit wary about the fact that the intergenic bases number is so high. I assume I messed up generating the list interval file.

I first aligned my yeast data with STAR, then ran Picard MarkDuplicates, and finally ran Picard CollectRnaSeqMetrics.

I generated the list interval file as follows:

GTF=Saccharomyces_cerevisiae.R64-1-1.86_transcripts.gtf
INPUT=Aligned.sortedByCoord.out.dedup.bam
LIST=interval.list

samtools view -H $INPUT > $LIST
grep "rRNA" $GTF | cut -s -f 1,4,5,7,9 >> $LIST

I only fed the rRNA information into the list file as suggested by this comment: A: Create Interval List for Picard CollectRNASeqmetrics

Anyone have any pointers on generating an appropriate list file?