A Robust, Streamlined, Enzyme-based DNA Library Preparation
Method Amenable to a Wide Range of DNA Inputs Style
Lynne Apone, Karen McKay, Vaishnavi Panchapakesa, Bradley Langhorst, Timur Shtatland, Chen Song, Pingfang Liu,Christine Sumner, Christine Rozzi, Melissa Arn, Daniela Munafo, Fiona Stewart, Eileen Dimalanta and Theodore Davis
New England Biolabs, Inc.
240 County Road, Ipswich, MA 01938, USA.

INTRODUCTIONULTRAULTRAII FS PRODUCES
THE HIGHEST
YIELD YIELDLIBRARIESII FS PRODUCES
THE HIGHEST

In order for Precision Medicine to be widely implemented, robust, accurate, methods
to process and interrogate large numbers of human samples are required. Whileadvances in Next Generation sequencing (NGS) technologies have positioned it at
the forefront of the Precision Medicine Initiative, it is ideally suited for deep molecularanalysis of a single sample, rather than the processing and interrogation ofthousands. In order to overcome this limitation, we have developed a robust,streamlined library construction method that integrates enzyme-based DNA
fragmentation with end repair and dA-tailing. This method utilizes a single protocol fora wide range of DNA input amounts and types. In addition, it eliminates the need for
expensive equipment to fragment DNA and numerous cleanup and liquid transfersteps, reducing time, cost and errors associated with library construction

Libraries were prepared from Human NA19240 genomic DNA using the input amounts andnumbers of PCR cycles shown. For NEBNext Ultra II FS, a 20-minute fragmentation time
was used. For Kapa™ HyperPlus libraries, input DNA was cleaned up with 3X beads prior tolibrary construction, as recommended, and a 20-minute fragmentation time was used.
Illumina® recommends 50 ng input for Nextera, and not an input range; therefore, only 50 ng
was used in this experiment. “Covaris®” libraries were prepared by shearing each input
amount in 1X TE Buffer to an insert size of ~200 bp using a Covaris instrument, followed bylibrary construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Error
bars indicate standard deviation for an average of 3–6 replicates performed by two
independent users.

METHODSENZYME BASED FRAGMENTATION COMBINED WITHNEBNEXT® ULTRA™ II DNA LIBRARY PREPARATIONSUPERIOR GC COVERAGE WITH HUMAN AND MICROBIAL
SUPERIOR GC COVERAGE
GENOMES WITH HUMAN AND

MICROBIAL GENOMESAA) Libraries generated using the indicatedkits, 50ng of Human gDNA (NA19240),
and 5 cycles of PCR were sequenced onan Illumina MiSeq® (2 x 76 bp). B)
Libraries prepared from 1 ng of a genomicDNA
mix
containing
Haemophilus
influenzae,
Escherichia
coli(K-12MG1655),
andRhodopseudomonas
palustris were amplified for 9 cycles ofPCR and sequenced on an IlluminaMiSeq®. Reads generated in A and Bwere mapped using Bowtie 2.2.4 and GC
coverage information was calculated
using
Picard’s
CollectGCBiasMetrics(v1.117). Expected normalized coverageof 1.0 is indicated by the horizontal greyline, the number of 100bp regions at each
GC% is indicated by the vertical grey
bars, and the colored lines represent thenormalized coverage for each library.

IntactDNA

STEPSSingle tube

Fragment
End Repair
dA-Tail

AdaptorLigation

Clean-upSize SelectAmplify

Clean-up

Genomic DNA isolated from a variety of sources was used to construct Illumina libraries. DNA(100pg - 500ng) was fragmented, end repaired and dA-tailed in a single step followed byadaptor ligation in the same tube. PCR amplified libraries were sequenced, reads aligned to theappropriate reference genome, and quality metrics generated using Picard tools.

B

RESULTSULTRA II FS ENABLES TIME_DEPENDENT FRAGMENTATIONAA) 100 ng of Human NA19240 genomic
DNA was incubated with the NEBNext Ultra
II FS Enzyme Mix and Reaction Buffer for 5,10, 15, 20, 25, 30 and 40 minutes at 37 ̊C,
followed by 65 ̊C for 30 minutes. Afterclean-upusingNEBNext
SamplePurification Beads, size was assessedusing the Agilent Bioanalyzer. B) Libraries
were constructed using the NEBNext Ultra IIFS kit and 100 ng of Human NA19240
genomic DNA, with fragmentation times of5, 10, 15, 20, 25, 30 and 40 minutes, and 4
PCR cycles. Size selection was not
performed. After clean-up using NEBNext
Sample Purification Beads, size wasassessed using the Agilent Bioanalyzer.
NEBNext Ultra II FS shows expected final
librarysizes
consistentwiththefragmentation sizes seen in A).

B

ROBUST FRAGMENTATION INDEPENDENT OF DNA
AND BUFFERABNEBNEXT® ULTRA™ II FS DNA ENABLES ACCURATE VARIANT
DETECTIOULTRA II FS ENABLES ACCURATE VARIANT DETECTIONAB

Tru-Q 1 (5% Tier) Reference Standard (Horizon® HD728) was spiked into wild type human
DNA (NA19240) at varying expected allele frequencies. 100 ng DNA mix was used togenerate NEBNext® Ultra™ II FS libraries, followed by target enrichment using the ClearSeq®
Comprehensive Cancer XT panel (Agilent® #5190-8011). A) Droplet digital PCR (Bio-Rad®
QX200) detected 11 of the known Tru-Q 1 mutations in the input DNA sample as well as theUII FS libraries at expected frequencies. B) Comparison of observed variant allele frequency(VAF) detected from sequencing data with the expected VAF from the 0.5% EAF. Libraries
were sequenced on a MiSeq (2x75 bp) to an average depth of 2000x coverage. Reads werealigned with BWA-MEM (Li and Durbin, 2010) and duplicates were marked either by alignment
coordinates alone or alignment coordinates and UMIs. Variants were detected with Scalpel(Narzisi et al., 2014), Mutect (Cibulskis et al., 2013), and Vardict (Lai et al., 2016) andconfirmed by hand for low allele frequencies.

CONCLUSIONSA) Libraries were made using 100 ng of human genomic DNA and the NEBNext® Ultra™ II FS
kit or the Kapa™ HyperPlus Kit. Libraries were fragmented to generate 200 bp inserts (320 bplibraries) from DNA resuspended in H2O, 10mM Tris, 0.1X TE or 1X TE . Kapa™ HyperPlus
libraries were made using the recommended dilution of the supplied Conditioning Solution(CS), or using DNA in 10mM Tris, 0.1X TE or 1X TE, in the absence of either CS or 3X beadclean up. Library size distribution was assessed using the Agilent® Bioanalyzer®. B) Libraries
were prepared from human genomic DNA using the input amounts shown. NEBNext® Ultra™ II
FS libraries were prepared using a 20-min fragmentation time. For Kapa™ HyperPlus, therecommended bead clean-up was performed prior to the 20 min fragmentation. Library size
was assessed using the Agilent® Bioanalyzer®. Low input (1 ng and below) libraries wereloaded on the Bioanalyzer® without dilution. High input libraries were loaded with a 1:5 dilution
in 0.1X TE.

§ Ultra™ II FS enables high-quality library construction from a broad range of DNAquantities and qualities§ Sequencing metrics of FS libraries are similar or superior to those produced withmechanically sheared DNA§ Ultra™ II FS maintains mutation abundance in samples without introducing bias
§ These advances will allow greater use and adoption of NGS technologies in clinical anddiagnostic settings

REFERENCES
Li, H., and Durbin, R. 2010. Fast and accurate long-read alignment with Burrows–Wheeler transform.
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Narzisi, G., et al. 2014. Accurate de novo and transmitted indel detection in exome-capture data using
microassembly. Nature Methods 11:1033–1036.
Cibulskis, et al. 2013. Sensitive detection of somatic point mutations in impure and heterogeneous cancer
samples. Nature Biotechnology 31:213–219.
Lai, Z., et al 2016. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer
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