NEBNext Ultra II libraries provide superior results in exome enrichment
|SAMPLE 1||SAMPLE 2|
|Library Prep Kit||Ultra II||Kapa|| Ultra II
|Total Reads||81 M|| 81 M
|| 81 M
|| 81 M
|% 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.
This video walks you through DNA Library Preparation using the NEBNext Ultra II DNA Library Prep Kit. The video also includes tips for optimization as well as safe stopping points.
These 12 quick tips for NGS library preparation are a great refresher if you're a seasoned pro, or a great introduction if you're a beginner.