RNA-seq: concern about quantification of transcriptome

mlacencio · 10-26-2013, 12:53 PM

Hi everybody!

I'd like to generate and quantify whole transcriptomes by using RNA-seq in the near -- well, not so near -- future but I am completely newbie on this method.

My doubt concerns mainly the quantification capacity of RNA-seq: how can RNA-seq be used to quantify transcripts if a PCR amplification has to be done after the adapter ligation in the library preparation setp? How can this amplification step not impair the post-sequencing quantification of transcripts?

Is the amount of PCR-amplified products directly proportional to the initial amount of cDNAs in this case?

Thank you in advance!

Marcio

Chipper · 10-27-2013, 01:15 PM

There will be some sequence-specific bias in the amplification but usually one compares the same sequences (genes) under different conditions, and with fragmented samples which makes it less of a problem. It is possible to use molecular identifiers (random sequences) in the adaptors to account for PCR bias (google qRNA-seq or "Counting absolute number of molecules using unique molecular identifiers" for more info).

mlacencio · 10-27-2013, 05:15 PM

Hi Chipper! Thank you for your reply!

Actually my main concern is about the number of PCR cycles. It is known that the quantification of PCR products is unreliable if PCR reaches its saturation level. In general, how many cycles are allowed in the amplification step of a library prep for RNA-seq?

Thank you again!

benjamir · 10-31-2013, 01:11 PM

Hi Mlacencio,
The Illumina TruSeq RNA-seq protocol calls for 15 cycle (total RNA 0.1 ug - 4 ug optimized).
A recent single-cell method, Quartz-Seq, performs 21 cycles of whole transcriptome amplification PCR prior to 10 - 12 cycles of library PCR.

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10-26-2013, 12:53 PM	#1
mlacencio Member Location: Brazil Join Date: Oct 2013 Posts: 12	RNA-seq: concern about quantification of transcriptome Hi everybody! I'd like to generate and quantify whole transcriptomes by using RNA-seq in the near -- well, not so near -- future but I am completely newbie on this method. My doubt concerns mainly the quantification capacity of RNA-seq: how can RNA-seq be used to quantify transcripts if a PCR amplification has to be done after the adapter ligation in the library preparation setp? How can this amplification step not impair the post-sequencing quantification of transcripts? Is the amount of PCR-amplified products directly proportional to the initial amount of cDNAs in this case? Thank you in advance! Marcio

10-27-2013, 01:15 PM	#2
Chipper Senior Member Location: Sweden Join Date: Mar 2008 Posts: 324	There will be some sequence-specific bias in the amplification but usually one compares the same sequences (genes) under different conditions, and with fragmented samples which makes it less of a problem. It is possible to use molecular identifiers (random sequences) in the adaptors to account for PCR bias (google qRNA-seq or "Counting absolute number of molecules using unique molecular identifiers" for more info).

10-27-2013, 05:15 PM	#3
mlacencio Member Location: Brazil Join Date: Oct 2013 Posts: 12	Hi Chipper! Thank you for your reply! Actually my main concern is about the number of PCR cycles. It is known that the quantification of PCR products is unreliable if PCR reaches its saturation level. In general, how many cycles are allowed in the amplification step of a library prep for RNA-seq? Thank you again!

10-31-2013, 01:11 PM	#4
benjamir Junior Member Location: Houston, TX Join Date: Nov 2012 Posts: 4	Hi Mlacencio, The Illumina TruSeq RNA-seq protocol calls for 15 cycle (total RNA 0.1 ug - 4 ug optimized). A recent single-cell method, Quartz-Seq, performs 21 cycles of whole transcriptome amplification PCR prior to 10 - 12 cycles of library PCR.