NEBNext solutions for challenging methods & samples®

Brittany S. Sexton, Margaret R. Heider, Jian Sun, Louise Williams, Matthew Angel, Brad W.
Langhorst & Pingfang Liu| New England Biolabs, Inc.

NEBNext UltraShear® (NEB# M07634)

NEBNext UltraShear FFPE DNA Library Prep Kit (NEB# E6655)
A library prep workflow designed to improve the data output and sequencing
accuracy from FFPE DNA independent of sample quality

NEBNext UltraShear improves libraries for challenging methods & samples
compared to other fragmentation methods
5mC Library Prep:

FFPE DNA Library Prep:•

Higher library yields•

Improved sequencing metrics•

Improved sequencing metrics•

Reduced artificial mutations•

Improved CpG CoverageNEBNext UltraShear fragments high-quality gDNA in a time-dependent manner****New enzyme mixes optimized for FFPE library prep

• New and more efficient enzymatic DNA repair using NEBNext FFPE DNA Repair v2• New NEBNext UltraShear enzymatic fragmentation mix optimized for use with FFPE DNA

NA12878 DNA was fragmented for 5–45 minutes at37°C followed by 15 minutes at 65°C.
Fragmentation occurs during the 37°C incubation
step of NEBNext UltraShear. The average
fragmentation size and pattern (High Sensitivity
D5000 ScreenTape® on Agilent® TapeStation®) isbased on fragmentation time.

Improved library yields & sequencing metrics in EM-seq™ libraries produced
using NEBNext UltraShear

(A)

(C)

• New NEBNext MSTC™ FFPE PCR Master Mix to achieve high yields for target enrichment• 5 – 250 ng input of FFPE DNA required, validated on FFPE with DIN 1.5 to 6
• Compatible with high quality DNA for convenience in processing with matched high quality DNA

Use of NEBNext FFPE DNA Repair v2 upstream of NEBNext UltraShear
improves library yield and coverage
(A)

(B)

(B)

(D)

Libraries were prepared in duplicate from 50 ng of unsheared FFPE DNA ranging in quality from DIN 1.8 to DIN 6.8 using either theNEBNext UltraShear FFPE DNA Library Prep Kit or NEBNext UltraShear with the NEBNext Ultra II DNA Library Prep Kit. The NEBNext
FFPE DNA Library Prep Kit or the NEBNext Ultra II DNA Library Prep Kit were used to prepare libraries from the same FFPE DNA
samples sheared to 350 bp with the Covaris ME220 instrument. UMI-containing adaptors and 9 PCR cycles were used for all libraries,and final library yield was quantified using the Qubit High-Sensitivity dsDNA Assay (A). (B) FFPE DNA 1.8 libraries were then captured
using a custom panel designed with Twist Bioscience and sequenced on the NovaSeq6000 to 15M PE reads. Target enrichment quality
metrics were obtained using Picard HS Metrics (v. 2.18.29). The improved yield obtained with FFPE DNA Repair v2 and UltraShear(NEBNext UltraShear FFPE DNA Library Prep Kit) translates to improved complexity (higher mean target coverage) and improved
coverage uniformity (lower Fold-80 base penalty).

NEBNext UltraShear FFPE Library Prep Kit enables higher yields thancompetitor enzymatic fragmentation kits for low quality FFPE DNA
NA12878 DNA spiked with control DNA (CpG methylated pUC19 DNA and unmethylated lambda DNA) was fragmented byeither NEBNext UltraShear or Covaris® ME220 followed by EM-seq library preparation. Technical replicates were generated foreach input amount. All libraries were sequenced on the same flowcell of an Illumina® NovaSeq® 6000 (2 x 100 bases). 725
million reads were sampled from each library for methylation analysis. (A) Library yields were quantified using Agilent
TapeStation with the High Sensitivity D1000 ScreenTape. EM-seq libraries fragmented by NEBNext UltraShear have higher
yields than Covaris for the same number of PCR cycles for each input. (B) The read count of inserts at each size (bp) wasplotted, illustrating that NEBNext UltraShear and Covaris fragmentation resulted in comparable sequenced insert sizes. (C) The
GC distribution for NEBNext UltraShear and Covaris and across inputs for EM-seq libraries were plotted. The GC distributions
are similar in the 30% to 60% GC, which contains most of the genome. (D) NEBNext UltraShear and Covaris fragmentation
used ahead of the EM-seq workflow yielded a similar number of CpGs (~54 million) at minimum 1X coverage. At minimum 10X
coverage, more CpGs are identified when NEBNext UltraShear is used, due to improved library diversity and coverage
evenness.

The NEBNext UltraShear FFPE DNA Library Prep Kit
enables higher library yields than competitor library prepkits. Libraries were prepared in duplicate from 100 ng of lowquality, normal tissue FFPE DNA (DIN 1.8) and 9 PCRcycles, using the NEBNext UltraShear FFPE DNA LibraryPrep Kit. Results were compared to other enzymatic
fragmentation-based library prep kits that have beenvalidated for use with FFPE samples, using each vendor’s
own recommended adaptors Note: kits from Agilent andWatchmaker Genomics were not available for purchase.

Enzymatic Fragmentation and DNA repair improve library quality andsequencing accuracy

Similar methylation is detected by EM-seq with NEBNext UltraShear & Covaris
(A)
(A)

(B)

NA12878 DNA spiked with control DNA (CpG methylated pUC19 DNA and unmethylated lambda DNA) was fragmented byeither NEBNext UltraShear or Covaris ME220 followed by EM-seq library preparation. Technical replicates were generated foreach input amount. All libraries were sequenced on the same flowcell of an Illumina NovaSeq 6000 (2 x 100 bases). 725 million
reads were sampled from each library for methylation analysis. (A) The percent of aggregated methylation in all contexts aresimilar for EM-seq libraries fragmented by NEBNext UltraShear or Covaris. The spike-in control DNAs used in the EM-seq
workflow had expected methylation: CpG methylated pUC19 had > 97% methylated Cs in CpG context and < 1.5% methylated
Cs in other contexts; unmethylated Lambda had < 0.5% methylated Cs in all contexts (data not shown). (B) Approximately 53
million CpGs were common to all libraries at a 1X sequencing depth. There is high correlation between CpG methylation ofNEBNext UltraShear and Covaris EM-seq libraries for 200 and 10 ng inputs.

(A)

(B)

(C)

NEBNext UltraShear with FFPE DNA improves usable reads& reduces artificial mutations

FFPE DNA was fragmented using NEBNext UltraShear, Covaris
ME220, and other commercially available enzymatic
fragmentation methods. All samples were fragmented according
to the respective protocols. Fragmentation with NEBNext dsDNA
Fragmentase®, Kapa kits and the Agilent kit was followed by abead cleanup and library construction using the NEBNext Ultra II
DNA library Prep Kit for Illumina. NEBNext UltraShear andCovaris-sheared samples were followed directly by use of theNEBNext Ultra II DNA Library Prep Kit for Illumina, and NEBNext
Ultra II FS samples followed the recommended protocol for library
prep. Each library was sequenced using the Illumina NextSeq®
500. 2 million (2 x 76 base) reads were used for this analysis. (A)Percent of usable reads (mappable, proper pairs, and nonduplicates reads) were measured for each library and averaged
for technical replicates (bars represent error between two
technical replicates) for all fragmentation methods. FFPE DNA
libraries fragmented with NEBNext UltraShear had the highest
percent of usable reads and had similar percent usable reads as
high-quality DNA libraries (high-quality DNA had a comparable
percent of usable reads across all fragmentation methods ≥ 96%;
data not shown). (B & C) Artificial C to T mutations were
calculated with Tasmanian tool for read 1 and 2 and averaged fortechnical replicates (bars represent error between two technicalreplicates). The libraries fragmented with NEBNext UltraShear
resulted in the lowest C to T artificial mutation frequency
compared to other fragmentation methods for FFPE DNA bothreads (R1= Read 1 and R2= Read 2).

(B)

The NEBNext UltraShear FFPE DNA Library Prep Kit improves library quality and sequencing accuracy compared to competitor library
prep kits. (A) Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using
the NEBNext UltraShear FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kitsthat have been validated for use with FFPE samples, using each vendor’s own recommended adaptors. Libraries were sequenced on theIllumina NovaSeq 6000 (2 x 100 base reads) and library quality metrics were assessed using Picard Alignment Summary Metrics (version1.56.0). The level of foldback reads was calculated using Seq_frag_remap (version 0.2). The NEBNext UltraShear FFPE DNA LibraryPrep Kit improves library quality by reducing the percentage of unmapped, chimeric, non-properly paired, and foldback reads. (B) The
average frequency of C→T mutations at each C position (left) and G→T mutations at each G position (right) in Read 1 and 2 wascalculated for two technical replicates using Tasmanian (version 1.0.7). C→T mutations arising from cytosine deamination and G→Tmutations arising from oxidative damage in low quality FFPE DNA are effectively repaired by the NEBNext FFPE DNA Repair v2 Mix in
the NEBNext UltraShear FFPE DNA Library Prep Kit. Other kits show a high level of C→T and G→T artifacts in low quality FFPE DNA
due to a lack of DNA damage repair.

NEBNext UltraShear FFPE DNA Library Prep enables high on-target coverage inhybrid capture libraries
Libraries were prepared in duplicate from 100 ng of low quality,
normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using
the NEBNext UltraShear FFPE DNA Library Prep Kit. Results
were compared to other enzymatic fragmentation-based library
prep kits that have been validated for use with FFPE samples,with each vendor’s own recommended adaptors (IDT® xGen®
EZ UNI, Kapa EvoPlus® Library Prep Kit, QuantaBio® sparQ
DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit).
The full library yield was used in singleplex target enrichmentwith a custom cancer panel (Twist Bioscience) and libraries
were sequenced on the Illumina NovaSeq 6000 (2 x 100 basereads). 15 million paired-end reads were trimmed with Fastp(version 0.20.0) and mapped with BWA mem (version 0.7.17) tothe T2T reference. Duplicates were marked using Picard
MarkDuplicates (version 2.20.6). Target enrichment quality
metrics were assessed using Picard HS Metrics (version2.18.29). The improved yield, coverage, and fraction of usable
reads observed in NEBNext UltraShear FFPE DNA Library
Prep Kit whole genome sequencing (WGS) libraries correlates
to improved coverage, on-target rate, and coverage uniformity
in target enrichment libraries.