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Question: RNA-Seq High Expression Genes Lost
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gravatar for madkisson
3.5 years ago by
madkisson • 30
United States
madkisson • 30 wrote:

Hi

I'm noticing that after running my BAM files through CuffDiff and CuffNorm that some genes that I know to be very highly expressed [Alb in liver should be 1M-2M reads; Gapdh should be fairly highly expressed] are showing zero [Alb] to under 20 reads [Gapdh]. I find the expected sized pile-ups when i check my BAM files on IGV. I'm certain this must be a parameter that can be adjusted in the Cuff* tools and is a consequence of many of these tools not really being written for full transcriptome RNA-Seq analysis. Any suggestions on how to fix this?

Here's my set up:
Mouse total RNA sequenced with Illumina HiSeq
Fastq to BAM files using TopHat2; defaults for all settings
BAM files straight to 'CuffDiff For CummeRbund' and 'CuffNorm' for tabular outputs; 3 Test replicates/5 Reference replicates
Using iGenome UCSC mm10 gtf file for both CuffDiff and CuffNorm

Thanks
differential expression rnaseq cuffnorm cuffdiff • 1.2k views
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modified 2.5 years ago by ksriram • 0 • written 3.5 years ago by madkisson • 30
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gravatar for ksriram
2.5 years ago by
ksriram • 0
ksriram • 0 wrote:

In Cufflinks, you will often get an error message saying 'HIDATA' and an FPKM value of 0. You need to increase the max number of fragments per locus; by default this is set to 10^6, you could increase this to say 10^7

ADD COMMENT • link written 2.5 years ago by ksriram • 0
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