Hi All
I have come across an issue of 0 fpkm even when there are reads for a locus.
I downloaded mm10 genome and annotation from https://ccb.jhu.edu/software/tophat/igenomes.shtml as recommended.
I am using TopHat v2.1.1 and Cufflinks v2.2.1 for this analysis.
I have 2 technical replicates that have almost similar amount of reads and correlate well when it comes to protein coding genes (R=0.99, pearson) but some genes such as follows:
Genename REP1 REP2
Snord104 0 81982.5
Show 0 fpkm in one replicate but huge fpkm is another replicate. This is confusing because the amount of read counts for this locus is close enough in both replicates (REP1=154 reads and REP2=199 reads).
I have read forums that say how this is a bam file and annotation/gtf file sorting issue and has been fixed in the latest version and yet I am getting results like this.
Please help me understand what I am doing wrong here.
I will be extremely grateful for your concern.
Here is my command:
REF=/home/mparida/MM10_GENOME/Mus_musculus/UCSC/mm10/Sequence/WholeGenomeFasta/genome.fa
ANNOTATION=/home/mparida/MM10_GENOME/Mus_musculus/UCSC/mm10/Annotation/Archives/archive-2015-07-17-14-33-26/Genes/genes.gtf
BOWTIE2IDX=/home/mparida/MM10_GENOME/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome
REPEAT=/home/mparida/mm10_repeatmasker.gtf
cufflinks -o $ODIR/CUFFLINKS/ -p 16 -b $REF -u --library-type fr-unstranded -N --compatible-hits-norm -G $ANNOTATION -M $REPEAT $ODIR/Sample_2_REP1.bam
cufflinks -o $ODIR/CUFFLINKS/ -p 16 -b $REF -u --library-type fr-unstranded -N --compatible-hits-norm -G $ANNOTATION -M $REPEAT $ODIR/Sample_2_REP2.bam
Rocky
I have come across an issue of 0 fpkm even when there are reads for a locus.
I downloaded mm10 genome and annotation from https://ccb.jhu.edu/software/tophat/igenomes.shtml as recommended.
I am using TopHat v2.1.1 and Cufflinks v2.2.1 for this analysis.
I have 2 technical replicates that have almost similar amount of reads and correlate well when it comes to protein coding genes (R=0.99, pearson) but some genes such as follows:
Genename REP1 REP2
Snord104 0 81982.5
Show 0 fpkm in one replicate but huge fpkm is another replicate. This is confusing because the amount of read counts for this locus is close enough in both replicates (REP1=154 reads and REP2=199 reads).
I have read forums that say how this is a bam file and annotation/gtf file sorting issue and has been fixed in the latest version and yet I am getting results like this.
Please help me understand what I am doing wrong here.
I will be extremely grateful for your concern.
Here is my command:
REF=/home/mparida/MM10_GENOME/Mus_musculus/UCSC/mm10/Sequence/WholeGenomeFasta/genome.fa
ANNOTATION=/home/mparida/MM10_GENOME/Mus_musculus/UCSC/mm10/Annotation/Archives/archive-2015-07-17-14-33-26/Genes/genes.gtf
BOWTIE2IDX=/home/mparida/MM10_GENOME/Mus_musculus/UCSC/mm10/Sequence/Bowtie2Index/genome
REPEAT=/home/mparida/mm10_repeatmasker.gtf
cufflinks -o $ODIR/CUFFLINKS/ -p 16 -b $REF -u --library-type fr-unstranded -N --compatible-hits-norm -G $ANNOTATION -M $REPEAT $ODIR/Sample_2_REP1.bam
cufflinks -o $ODIR/CUFFLINKS/ -p 16 -b $REF -u --library-type fr-unstranded -N --compatible-hits-norm -G $ANNOTATION -M $REPEAT $ODIR/Sample_2_REP2.bam
Rocky
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