how to calculate the mean sequencing depth of RNA-seq

wangli · 11-02-2012, 10:06 AM

Dear users

I tried to calculate the mean sequencing depth of RNA-seq. Firstly, I utilized GATK to calculate that with the whole genome as reference. It turned out that some samples with around 15 million mapped reads only have around 2.5* mean sequencing depth. That looks to me weird. Some people suggested me to calculate it by hand, such as: (number of mapped reads * read length)/total length of the species' transcriptome

It looks reasonable. How do you calculate the sequencing depth of RNA-seq?
I will appreciate your comments very much!

woodydon · 02-18-2014, 06:38 PM

You should calculate the sequencing depth of a gene or an exon. It doesn't make sense to have a number representing the overall sequencing depth of RNA-Seq. DNA and RNA-Seq are different.

puggie · 02-19-2014, 03:56 PM

You could use e.g. Cufflinks or DEseq to calculate the library depths, at least that would provide you a scaling factor for inter-library comparisons and normalization. I think the "sequencing depth" as per DNA-seq is not comparable in this respect at all.

Jeremy · 02-19-2014, 05:44 PM

The most informative coverage metric for RNA-seq is the number of genes with x number of reads, e.g. 80% of the known genes had at least 10 reads mapping to them, 95% of the known genes had at least one read map.

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11-02-2012, 10:06 AM	#1
wangli Member Location: Texas Join Date: Apr 2012 Posts: 48	how to calculate the mean sequencing depth of RNA-seq Dear users I tried to calculate the mean sequencing depth of RNA-seq. Firstly, I utilized GATK to calculate that with the whole genome as reference. It turned out that some samples with around 15 million mapped reads only have around 2.5* mean sequencing depth. That looks to me weird. Some people suggested me to calculate it by hand, such as: (number of mapped reads * read length)/total length of the species' transcriptome It looks reasonable. How do you calculate the sequencing depth of RNA-seq? I will appreciate your comments very much!

02-18-2014, 06:38 PM	#2
woodydon Member Location: https://www.researchgate.net/profile/Woody_Lin Join Date: Jan 2010 Posts: 52	You should calculate the sequencing depth of a gene or an exon. It doesn't make sense to have a number representing the overall sequencing depth of RNA-Seq. DNA and RNA-Seq are different.

02-19-2014, 03:56 PM	#3
puggie Member Location: Europe Join Date: Nov 2011 Posts: 52	You could use e.g. Cufflinks or DEseq to calculate the library depths, at least that would provide you a scaling factor for inter-library comparisons and normalization. I think the "sequencing depth" as per DNA-seq is not comparable in this respect at all.

02-19-2014, 05:44 PM	#4
Jeremy Senior Member Location: Pathum Thani, Thailand Join Date: Nov 2009 Posts: 190	The most informative coverage metric for RNA-seq is the number of genes with x number of reads, e.g. 80% of the known genes had at least 10 reads mapping to them, 95% of the known genes had at least one read map.