Uniform GC Coverage
Libraries from varying input amounts of three microbial genomic DNAs with low, medium and high GC content (H. influenza, E. coli and H. palustris) were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit. In all cases, uniform coverage was obtained, regardless of GC content and input amount (A). GC coverage of libraries prepared using other commercially available kits was also analyzed using the same trio of genomic DNAs. Again, NEBNext Ultra II provided good GC coverage (B).
Ultra II libraries provide the highest quality sequencing data
|DNA INPUT||LIBRARY KIT||TOTAL READS||% MAPPED||% DUPLICATION||% CHIMERAS|
|100 ng||Ultra II||419,093,838||96||1.87||0.48|
|1 ng||Ultra II||226,860,968||96||3.96||0.44|
Libraries were prepared from Human NA19240 genomic DNA using the input amounts and library prep kits shown, following manufacturers’ recommendations. Libraries were sequenced on the Illumina NextSeq 500. Reads were mapped to the GRCh37 reference using Bowtie 2.2.4. This data illustrates that the NEBNext Ultra II DNA Library Prep Kit enables high quality sequence data, even with very low input amounts.
% Mapped: The percentage of reads mapped to Human GRCh37 reference.
% Duplication: The percentage of mapped sequence that is marked as duplicate.
% Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to different chromosomes.
Coverage of Known Low-Coverage Regions of the Human Genome
Regions of the human genome typically covered at a relatively low level have been identified (2), and the majority of these regions have high GC content. Library preparation can contribute to low and uneven sequence coverage, or even drop-outs, of these challenging regions. Depending on the polymerase used, PCR amplification of a library can result in under-representation of GC-rich regions, and libraries constructed by PCR-free workflows can provide more uniform coverage than amplified libraries (1). Improvements in efficiency and reduction in bias at each step in library preparation, including improved uniformity of library amplification over the full range of GC content improves the evenness of sequence coverage of these regions. Here we show a comparison of sequencing data from human genomic DNA libraries prepared with NEBNext Ultra II and other commercially available kits. Ultra II provided the highest and most uniform coverage of difficult sequence regions, as well as the coverage most similar to the PCR-free library (see below).
As described above, an ideal library will represent completely and proportionally the sequence of the input DNA. When library preparation is inefficient or when input amounts for a library are very low, there is a risk that the resulting library will lack this diversity, and that some sequences will be over- or under-represented. Comparison of the level of sequence coverage, in 10 kb intervals, achieved with libraries from different input amounts is a useful measure to determine the effect of input amounts on coverage. The increased efficiency of each step in the NEBNext Ultra II library workflow improves the library diversity. Here we show comparisons of libraries prepared for 100 ng, 1 ng and 500 pg human genomic DNA prepared using NEBNext Ultra II. The results demonstrate consistently even coverage for the range of input amounts indicated (see below).
- Kozarewa, I. et al. (2009). Amplification-free Illumina sequencing – library preparation facilitates improved mapping and assembly of (G+C) – biased genomes. Nat. Methods 6:291–295.
- Aird, D. et al. (2011). Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biology 12(2), R18.
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.