TruSeq PCR-free libraries inefficient ligation of adapters

kgoglin · 11-30-2018, 12:51 PM

It seems to be well known that the ligation efficiency of the adapters for the TruSeq PCR-free library prep is low. Bioanalyzer concentrations can be 3-30 fold higher than qPCR. I believe the qPCR concentrations are accurate, however, the sequencing runs are under-clustered. I suspect the libraries are not getting denatured properly. Has anyone had this happen?

My theory is that the NaOH is being used up on the DNA fragments with no adapters and therefore less NaOH is available to denature the libraries.

Thanks,
Karrie

thermophile · 01-08-2019, 02:00 PM

what kind of samples? I'm fighting with difficult really high gc bacteria that I've truseq pcr free prepped. Ive started heat denaturing after NaOH denaturing. it may be helping a bit? I'm still testing the libraries by spiking them in other runs, haven't tried this on its own run yet

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Doing PCR enrichment on PCR-free TruSeq lib?	JBKri	Sample Prep / Library Generation	2	10-08-2015 08:00 AM
TruSeq PCR-free, ligate adapters to amplicons?	JBKri	Sample Prep / Library Generation	2	06-05-2015 10:47 AM
TrueSeq PCR free POOR LIGATION efficiencies	Degraz	Illumina/Solexa	3	04-08-2015 06:03 AM
Illumina TruSeq PCR-Free - Low Final Concentration WGS Rat DNA Libraries	agileta	Illumina/Solexa	1	11-18-2014 08:11 PM
PCR-Free Libraries & Bioinformatic PCR Duplicate detection?	aeonsim	Bioinformatics	3	10-23-2013 02:36 PM

11-30-2018, 12:51 PM	#1
kgoglin Member Location: San Diego Join Date: Dec 2014 Posts: 15	TruSeq PCR-free libraries inefficient ligation of adapters It seems to be well known that the ligation efficiency of the adapters for the TruSeq PCR-free library prep is low. Bioanalyzer concentrations can be 3-30 fold higher than qPCR. I believe the qPCR concentrations are accurate, however, the sequencing runs are under-clustered. I suspect the libraries are not getting denatured properly. Has anyone had this happen? My theory is that the NaOH is being used up on the DNA fragments with no adapters and therefore less NaOH is available to denature the libraries. Thanks, Karrie

01-08-2019, 02:00 PM	#2
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 237	what kind of samples? I'm fighting with difficult really high gc bacteria that I've truseq pcr free prepped. Ive started heat denaturing after NaOH denaturing. it may be helping a bit? I'm still testing the libraries by spiking them in other runs, haven't tried this on its own run yet __________________ Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.