NEBNext DNA Library Prep Master Mix Set for Illumina from New England BioLabs

January 30, 2013

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Medical University Graz
Institute of Human Genetics
Post Doc

Comparison of The NEBNext DNA Library Prep Master Mix Set for Illumina (NEB) and the TruSeq Sample Preparation Kit (Illumina). The NEBnext library protocol works as least as good as the Illumina protocol. Both library kits generated high quality libraries with an average size of 300-400 and high yield.

Company:

New England BioLabs

Product Name:

NEBNext® DNA Library Prep Master Mix Set for Illumina®

Catalog Number:

E6040S

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Almost all available next generation sequencing (NGS) platforms use the same principle for template preparation which includes random fragmentation of target DNA and addition of platform-specific adapter sequences to flanking ends. However, the quality of the prepared library is critical for obtaining high-quality data, no matter whether you perform shotgun-sequencing or sequencing of enriched targets. The NEBNext DNA Library Prep Master Mix Set for Illumina is a kit for preparing shotgun libraries for subsequent targeted enrichment or direct sequencing on an Illumina HiSeq or MiSeq. The kit includes all necessary reagents for end repair, A-tailing and adaptor ligation. However, adaptor must be purchased separately. The workflow is quite the same as with other shotgun-library protocols and takes a few hours.

Experimental Design and Results Summary

Application

Target enrichment and NGS

Starting Material

DNA from HCT116 and HT29 colon cancer cell lines. The DNA from the cell lines was isolated with Chemagen DNA Kit (Perkin Elmer) or QIAamp DNA Micro Kit (Qiagen). Both Kits provided high yields and high-quality DNA. DNA was quantified using a Nanodrop and 1µg of the DNA was sheared to approximately 300bp using the Covaris system.

Protocol Overview

We tested this kit and compared it to the TruSeq Sample Preparation Kit from Illumina. The libraries were prepared according to the manufacturer’s instructions using either the NEBNext kit or the TruSeq kit. Briefly, DNA was fragmented by sonication to a maximum of 300bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific aAdaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 10 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA7500 or High Sensitivity (See Figure). 500ng of each library were then pooled for a targeted enrichment of 370 cancer-associated genes using the TruSeq Enrichment trial kit (Illumina). This library pool, consisting of two enriched HT29 libraries (NEBNext and TruSeq, see Figure) as well as two enriched HCT116 libraries (NEBNext and TruSeq), was sequenced on an Illumina MiSeq.

Tips

Since enzymatic fragmentation (i.e. NEBNext® dsDNA Fragmentase®) never seems to be as random as the mechanical methods, I would recommend using the Covaris system for DNA fragmentation. We used AMPure XP Beads for all cleaning steps as well as for the size selection, which I highly recommend. However, in case of low quality samples (i.e. FFPE) or remaining adaptor dimers after PCR enrichment, I would suggest a gel- based size selection.

Results Summary

Both library kits generated high quality libraries with an average size of 300-400bp with a small range which is optimal for a 150bp paired end sequencing run an Illumina HiSeq. Both library preparations provided high yield and were directly entered into targeted enrichment. All 4 libraries achieved similar amounts of reads with very high quality score resulting in an average of 150x coverage for all samples. All variants found in the TruSeq libraries were also found in the NEBNext libraries. The accuracy of sequencing demonstrated the high quality of NGS libraries constructed with both kits.

Additional Notes

None

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Comparison of The NEBNext DNA Library Prep Master Mix Set for Illumina (NEB) and the TruSeq Sample Preparation Kit (Illumina). The NEBnext library protocol works as least as good as the Illumina protocol. Both library kits generated high quality libraries with an average size of 300-400 and high yield.

Summary

The Good

An advantage of the NEBNext is that you can order all modules of the kit separately in case some reagents are used up. Furthermore, these modules offer the ability to customize sample preparation. Furthermore, NEBNext is a little less expensive per reaction compared to the TruSeq Kit.

The Bad

Oligonucleotides are not included in the kit.

The Bottom Line

Taken together, we have found that the NEBNext library protocol is highly compatible with the Illumina chemistry. The library protocol works as least as good as the Illumina protocol.

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