Looking for a standardized method of library quantification and quality control

grawfin · 08-23-2019, 02:37 AM

Hello all,

I am wondering if anyone out there has discovered/created a standardized method for library quantification and quality control that works for them.

The issue that I keep running up against is that, depending on the method used for library preparation, the values given by tapestation, qbit, qpcr and fragment analyzer can vary wildly. Sometimes, for well prepared libraries I will get good consensus between the instruments. But when I don't, it turns quantification into a completely heuristic process.

I have observed the following trends:
10x libraries are better quantified on tapestation than qubit,
Amplicons and Nextera libraries don't seem to work well on tapestation OR qubit,

fragment analyzer and qpcr are both time-expensive, and the tapestation is just expensive.

With all that said, has anyone out there been able to completely standardize the process of QC and library quant for all library types? Thank you in advance.

Johnwang · 08-23-2019, 07:57 AM

We have NextSeq500/550, I have tried many lib quantification methods (Qubit, NanoDrop, tapestation, Quantfluor and qPCR), the only one works to me is qPCR with kit from KAPA. I have run many libraries constructed with different commercial kits, lab-protocols as well, all work with good cluster density and quality.

GW_OK · 08-23-2019, 09:12 AM

Something (Tape/BioA/FragA) for size estimation followed by KAPA qPCR for absolute quantification. That's the gold standard in my opinion. If it hasn't been qPCR'd we don't run it!

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Quality Control Library using QUBIT... help me!	soleulloa	Ion Torrent	1	09-01-2011 07:18 AM
DNA isolation & library quality control	Nak	Sample Prep / Library Generation	2	05-25-2011 01:39 PM
mRNA-seq library quality control	shilh	Illumina/Solexa	2	04-23-2010 07:09 AM
3'UTR library or random primed cDNA library for quantification?	Rosanne82	Sample Prep / Library Generation	0	06-26-2009 06:27 AM

08-23-2019, 02:37 AM	#1
grawfin Junior Member Location: Zurich, Switzerland Join Date: Jul 2019 Posts: 2	Looking for a standardized method of library quantification and quality control Hello all, I am wondering if anyone out there has discovered/created a standardized method for library quantification and quality control that works for them. The issue that I keep running up against is that, depending on the method used for library preparation, the values given by tapestation, qbit, qpcr and fragment analyzer can vary wildly. Sometimes, for well prepared libraries I will get good consensus between the instruments. But when I don't, it turns quantification into a completely heuristic process. I have observed the following trends: 10x libraries are better quantified on tapestation than qubit, Amplicons and Nextera libraries don't seem to work well on tapestation OR qubit, fragment analyzer and qpcr are both time-expensive, and the tapestation is just expensive. With all that said, has anyone out there been able to completely standardize the process of QC and library quant for all library types? Thank you in advance.

08-23-2019, 07:57 AM	#2
Johnwang Member Location: NE, USA Join Date: Jun 2016 Posts: 17	We have NextSeq500/550, I have tried many lib quantification methods (Qubit, NanoDrop, tapestation, Quantfluor and qPCR), the only one works to me is qPCR with kit from KAPA. I have run many libraries constructed with different commercial kits, lab-protocols as well, all work with good cluster density and quality.

08-23-2019, 09:12 AM	#3
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	Something (Tape/BioA/FragA) for size estimation followed by KAPA qPCR for absolute quantification. That's the gold standard in my opinion. If it hasn't been qPCR'd we don't run it!