Question

To find RPKM in RNA seq you first add up your total reads in a gene and divide by your scaling factor, a number decided by the scientist based on intended sequencing depth. The next step is to divide then divide by the length of the gene in kilobases. If the scaling factor was 1,000,000 this gives you RPKM or reads per kilobase million.

Is this correct? What does RPKM represent/ what kind of practical information does it give us? What is the scaling factor? Why do you divide the total reads by it? What is sequencing depth? Why would a higher scaling factor give us a higher sequencing depth?

Answer 1

Answer:

• The given steps above are correct. RPKM (Reads Per Kilobase Million) are found as following:

1. 1st one need to count up the Total Reads in the given sample and then divide this with the scaling factor.
2. This number is 1,000,000 this is called as the "per million" scaling factor.. By dividing to this value one obtains the sequencing depth giving the Reads Per Million(RPM)
3. By further division of RPM values with the length of the given gene(in kilobases) gives the RPKM value.

• RPKM : Reads Per Kilobase Million : It is made for the single end RNA-seq, where all the reads corresponds to the single fragment which is sequenced.
• The scaling factor is 1,000,000 which is used to divide the total reads so that one obtains the Reads Per Million values of the given sample.
• This division is a normalized unit of the transcript expression. It helps in scaling the transcript length to compensate as most of the RNA-seq will create more of the sequencing reads from the longer RNA molecules.
• Sequencing depth is mainly used for exome or the genome sequencing and it mainly refers to the number of reads that are covering each positions. This is otherwise called the Coverage.