NEBNext vs Illumina TruSeq ChIP-Seq library prep

khata · 08-04-2013, 05:55 AM

Hi, I was wondering if anyone did a comparison between ChIP-Seq data obtained through NEBnext and Illumina Truseq library prep.

We're seeing huge differences between the two kits (reproducible for two different transcription factors), in particular very low enrichment in high GC regions when using Illumina reagents. The problem is most visible at TSSs, where we see a dip in coverage.

Attaching some plots to illustrate it, ChIP1 and ChIP2 are two different transcription factors, IL is Illumina, NEB NEBnext. The venns show peak-level overlaps, the histograms GC content/peak, the lineplots mean extended read count at TSSs.

sara_ahmed · 08-07-2013, 09:14 AM

What was the enzyme used for library aplification? It's probably a result of the difference in the enzyme used for amplification that's giving you variable coverage of GC regions.

Sara Ahmed, PhD | Director of Sequencing | Cofactor Genomics
3141 Olive St. | St. Louis, MO 63103 | tel. (314) 531-4647

Jweber · 08-12-2013, 06:17 AM

@ sara_ahmed: The polymerases used in the kits are Phusion for NEBnext and a proprietary enzyme for Illumina TruSeq.

Hybridizationx · 08-21-2013, 11:50 AM

You should probably use NEBNext Hi-fidelity PCR mastermix as it is significantly better than Phusion or Illumina enzyme in our hands...also the same price as Phusion too

gwilkie · 08-22-2013, 11:28 AM

We've had good results with KAPA Hifi polymerase, its supposed to have very low amplification bias. Phusion is known to have poor amplification of high GC DNA.

BioGenomics · 09-16-2014, 08:38 PM

Hi all,

so is there a consensus on using NEB or Illumina ChipSeq lib prep kits ? I assume the NEB is way cheaper anyway, but seemingly had better performance from the latest post ?

thanks

Greg

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09-16-2014, 08:38 PM	#6
BioGenomics Member Location: Belgium Join Date: Apr 2009 Posts: 24	NEB or Illumina ? Hi all, so is there a consensus on using NEB or Illumina ChipSeq lib prep kits ? I assume the NEB is way cheaper anyway, but seemingly had better performance from the latest post ? thanks Greg

08-07-2013, 09:14 AM	#2
sara_ahmed Member Location: St Louis Join Date: Nov 2011 Posts: 13	What was the enzyme used for library aplification? It's probably a result of the difference in the enzyme used for amplification that's giving you variable coverage of GC regions. Sara Ahmed, PhD \| Director of Sequencing \| Cofactor Genomics 3141 Olive St. \| St. Louis, MO 63103 \| tel. (314) 531-4647

08-12-2013, 06:17 AM	#3
Jweber Junior Member Location: Switzerland Join Date: Feb 2012 Posts: 1	@ sara_ahmed: The polymerases used in the kits are Phusion for NEBnext and a proprietary enzyme for Illumina TruSeq.

08-21-2013, 11:50 AM	#4
Hybridizationx Junior Member Location: North America Join Date: Aug 2012 Posts: 2	You should probably use NEBNext Hi-fidelity PCR mastermix as it is significantly better than Phusion or Illumina enzyme in our hands...also the same price as Phusion too

08-22-2013, 11:28 AM	#5
gwilkie Member Location: Glasgow Join Date: Dec 2011 Posts: 27	We've had good results with KAPA Hifi polymerase, its supposed to have very low amplification bias. Phusion is known to have poor amplification of high GC DNA.