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Library Preparation for Target Enrichment Workflows

For hybridization-based target enrichment workflows the amount of library required can be large, generally in the microgram range. This can be challenging to achieve, especially when only small amounts of the original sample are available. NEBNext® Ultra™ II reduces the number of PCR cycles required to yield 1 μg or more of library, thereby enabling the use of low nanogram sample input amounts for library construction for target enrichment workflows (see below).
Number of PCR cycles required to generate ≥ 1 μg amplified library for target enrichment.
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Libraries were prepared from Human NA19240 genomic DNA using NEBNext Ultra II and the input amounts shown. Yields were measured after each PCR cycle and the number of cycles required to generate at least 1 μg of amplified library determined. Cycle numbers for Kapa™ Hyper were obtained from Kapa Biosystems website and plotted alongside the cycle numbers obtained experimentally for Ultra II.
Importantly, in addition to being of sufficient quantity, libraries must be of high quality to enable effective target enrichment and generation of high quality sequence data. Libraries were generated using NEBNext Ultra II and other commercially available library preparation reagents, followed by exome capture. Measurement of yields and sequencing metrics indicated superior performance from Ultra II libraries (see below).

NEBNext Ultra II provides superior yields for target enrichment applications

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For Nimblegen® enrichment, libraries were prepared with 100 ng of Human HG02922 genomic DNA, using NEBNext Ultra II with the NEBNext Adaptor and index primers, or the Kapa Library Preparation Kit Illumina Platforms with the SeqCap® Adapter Kit (Roche). Target enrichment was performed with NimbleGen SeqCap Human Exome v3.

For SureSelect XT enrichment, libraries were prepared with 200 ng of Human HG02922 genomic DNA, using NEBNext Ultra II or Kapa Hyper with the NEBNext Adaptor and index primers, or the SureSelect XT Reagent Kit, Illumina (ILM) platforms with Herculase® II Fusion DNA Polymerase. Target enrichment was performed with SureSelect® Human All Exon V6.

Manufacturers’ recommendations were followed, including use of the recommended number of PCR cycles for pre- and post-capture. NEBNext Ultra II libraries resulted in the highest post-capture yields for both enrichment workflows.
NEBNext Ultra II libraries provide superior results in exome enrichment
  SAMPLE 1 SAMPLE 2
 Library Prep Kit  Ultra II Kapa   Ultra II
 Kapa 
 Total Reads  81 M  81 M
  81 M
  81 M
 % Duplication  9.2  14.7  9.7  12.4
 % Selected Base  90.3  89.0  89.7  89.0
 Mean Target Coverage (x)  54  50  53  51
 % Zero Coverage  0.37  0.37  0.10  0.11
 Fold 80 Base Penalty  2.56  2.77  2.65  2.81
 HS Library Size (molecules)  167.9 M
  98.2 M
  156.1 M
  117.7 M

Pre-capture libraries were prepared with 100 ng of Human HG00096 (Sample 1) and HG02922 (Sample 2) genomic DNA, using NEBNext Ultra II with the NEBNext Adaptor and index primers, or the Kapa Library Preparation Kit Illumina Platforms with the SeqCap Adapter Kit (Roche). Hybrid selection of the human exome was performed with 1 μg of each library and SeqCap human exome v3 following the NimbleGen SeqCap protocol. Post-capture amplification was conducted with NEBNext Ultra II Q5 Hot Start Master Mix for Ultra II libraries, and Kapa HiFi Ready Mix for Kapa librar ies. Libraries were pooled and sequenced on an Illumina NextSeq 500 instrument. Sequencing reads were mapped to human GRCh37 reference and analyzed using Picard HS Metrics tool.

% Duplication: The percentage of mapped sequence that is marked as duplicate.
% Selected Base: On+Near Bait Bases/PF Bases Aligned.
Mean Target Coverage: The mean coverage of targets that received at least coverage depth = 2 at one base.
% Zero Coverage: The number of targets that did not reach coverage=2 over any base.
Fold 80 Base Penalty: The fold over-coverage necessary to raise 80% of bases in “non-zero-coverage” targets to the mean coverage level in those targets.
HS Library Size: The estimated number of unique molecules in the library.

The NEBNext Ultra II Kit leads to lower duplication rate, higher percentage of selected base, lower Fold 80 Base Penalty, and significantly larger HS library size, suggesting a higher degree of uniformity of NEBNext Ultra II libraries.

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