Streamlined Express DNA Library Preparation Methods
Meet Requirements of High-throughput Library Construction
Karen R. McKay, Chen Song, Kaylinnette Pinet, Matthew Campbell, Bradley W. Langhorst, V K Chaithanya Ponnaluri,
Pingfang Liu, and Keerthana Krishnan
New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.

INTRODUCTION
As next generation sequencing technologies improve and capacities expand, library construction has increasingly become the bottleneck for fast data turnaround and overall cost. There is agrowing need for faster, easier, and automatable protocols that perform reliably and do not compromise the quality of the libraries produced. We have developed two new NEBNext UltraExpress™
workflows with mechanical or enzymatic shearing approaches, allowing for a single tube, standardized protocol for fast library preparation. The streamlined, user-friendly workflows reduce handson time by combining reaction steps, removing clean-ups, and simplifying the protocols with a single adaptor concentration and one PCR cycling condition. They allow many DNA samples withvarying sources and input amount (10 ng to 200 ng) to be processed in one setting without normalization or adjusting reaction conditions. We have applied these two robust workflows to generatehigh quality sequencing data. We expect these library preparation methods will serve a myriad of NGS applications, including clinical and diagnostic settings.

NEBNext UltraExpress FS DNA library preparation
Fragment
End Repair
dA-Tailing

Intact DNA(10 – 200 ng)

ONE[adaptor]ONEcycle #Adaptor
Ligation

PCR
Amplify

NEBNext UltraExpress DNA library preparation

Fragmented DNA(10 – 200 ng)

Clean-up

End Repair
dA-TailingONE[adaptor]

ONEcycle #Adaptor
Ligation

PCRAmplify

6 cycles8 cyclesNEBNext UltraExpress FS DNA Library Prep Kit provides consistent
fragmentation and robust library yields over a wide input range(A) Library profile

Clean-up

NEBNext UltraExpress DNA Library Prep Kit produces robust library yields
and superior data quality(B) Library YieldInput (ng)50(B) GC Coverage(C) Insert Size

100 200

DNA Size (bp)10(A) Library Yield(A) Libraries made using 10 ng to 200 ng a 9:1 Human NA19240 genomic DNA (Coriell Institute for Medical Research) andEscherichia coli gDNA (Lofstrand Labs Limited) mixed sample, using the NEBNext UltraExpress FS DNA single-protocol workflow(e.g., same adaptor amount and 6 PCR cycles for all input amounts) showed uniform library profiles. (B) Libraries prepared intriplicate from 10, 50, 100 and 200 ng of a 9:1 Human NA19240 genomic DNA and Escherichia coli gDNA mixed sample, using theNEBNext UltraExpress FS DNA single-protocol workflow. Yields exceeded the minimum requirement (40 ng) for a single Illumina®
NovaSeq® 6000 run to achieve whole genome sequencing with at least 30X coverage.

Libraries were prepared from 10, 50, 100 or 200 ng of Human NA19240 genomic DNA (Coriell Institute for Medical Research) using
the same adaptor amount and 8 PCR cycles. (A) Yields exceeded the minimum requirement (40 ng) for a single Ilumina NovaSeq
6000 run to achieve whole genome sequencing with at least 30X coverage. Libraries were pooled and sequenced on an Illumina
MiSeq (2 x 75 bases). Data showed consistent GC coverage (B) and insert size (C). 2 million paired-end reads from each librarywere sampled (seqtk v1.3), adapter-trimmed (seqprep v0.1) and mapped to the GRCh38 reference (bowtie2 v2.4.5), and GC
coverage information was calculated using Picard's CollectGCBiasMetrics (v 1.56.0). In (B), the horizontal grey line indicates theexpected normalized coverage of 1.0, and the dots in shades of green represent read numbers at each GC%. The grey area plot is ahistogram representing the distribution of GC content in 100 bp windows of the reference genome.

NEBNext UltraExpress FS DNA Library Prep Kit produces libraries withuniform GC coverage and insert size from a range of input amounts

NEBNext UltraExpress DNA Library Prep Kit produces even GC coverage andinsert size for a range of sample types(A) GC Coverage(B) Insert Size(A) GC Coverage

Libraries were prepared from 10, 50, 100 and 200 ng of a 9:1 Human NA19240 genomic DNA (Coriell Institute for MedicalResearch) and Escherichia coli gDNA (Lofstrand Labs Limited) mixed sample, using the NEBNext UltraExpress FS DNA singleprotocol workflow (e.g., same adaptor amount and 6 PCR cycles for all input amounts). Libraries were pooled and sequenced on an
Illumina NextSeq® 500/550 (2 x 75 bases). Data showed consistent (A) GC coverage and (B) insert size. 2 million paired-end reads
from each library were sampled (seqtk v1.0), adaptor-trimmed (seqprep v0.1) and mapped to a composite reference containing
GRCh38 and E. coli MG1655 contigs (bowtie2 v2.5.0). GC coverage and insert size distributions were calculated using Picard's
CollectGCBiasMetrics and Picard CollectInsertSizeMetrics (v1.56.0); Picard CollectGCBiasMetrics. (1.56) was run on humanautosomes only due to the even copy number assumption of the tool. In (A), the horizontal grey line indicates the expected
normalized coverage of 1.0, and the dots in shades of green represent read numbers at each GC%. The grey area plot is ahistogram representing the distribution of GC content in 100 bp windows of the reference genome.

NEBNext UltraExpress FS DNA Library Prep Kit produces representative
GC coverage and insert size for a range sample types(A) GC Coverage(B) Insert Size(B) Insert Size

Libraries were prepared using a single protocol from cell-free DNA (cfDNA, 12 ng) without shearing and Maize DNA (10 ng and 100ng) sheared to 200bp (Covaris ME220). Libraries were pooled and sequenced on an Illumina MiSeq (2 x 75 bases). 1.4 million
paired-end reads from each library were sampled (seqtk v1.3), adapter-trimmed (seqprep v0.1), and aligned to either the GRCh38
reference (human) or Zea mays reference genome (maize) (bowtie2 v2.4.5). (A) All sample types showed representative GC
coverage. High and low input Maize DNA generated consistent GC coverage. The horizontal grey line indicates the expected
normalized coverage of 1.0, and the colored dots represent read numbers at each GC%. The grey area plot is a histogram
representing the distribution of GC content in 100 bp windows of the reference genome. (B) cfDNA insert size had the characteristic
fragmentation pattern with 167 bp peak size and periodicity feature of nucleosome-bound DNA in shorter fragments. 10 ng and 100ng Maize DNA showed consistent insert size, as expected with the shearing protocol used.

NEBNext UltraExpress DNA Library Prep Kit provides robust librarycomplexity(A) ZymoBIOMICS Microbial Community DNA Standard(B) ZymoBIOMICS Microbial Community DNA Standard II

Libraries were prepared using the NEBNext UltraExpress FS DNA protocol for 10 ng and 100 ng of genomic DNA from Haemophilus
influenzae, Escherichia coli, Rhodopseudomonas palustris and Bordetella pertussis. Data shown (A) representative GC coverageand (B) insert size peaks across samples with genome GC contents of 38%-68% GC. Libraries were pooled and sequenced on an
Illumina NextSeq 500/550 (2 x 75 bases). 2 million paired-end reads from each library were sampled (seqtk v1.0), adaptor-trimmed(seqprep v0.1), and aligned to their respective reference genomes (bowtie2 v.2.5.0). GC coverage and insert size distributions werecalculated using Picard's CollectGCBiasMetrics and Picard CollectInsertSizeMetrics (v1.56.0). The horizontal grey line indicates theexpected normalized coverage of 1.0, and the colored dots represent read numbers at each GC%. The grey area plot is a histogram
representing the distribution of GC content in 100 bp windows of the reference genome for the 10 and 100 ng inputs.

NEBNext UltraExpress FS DNA Library Prep Kit provides robust librarycomplexity(A) ZymoBIOMICS Microbial Community DNA Standard(B) ZymoBIOMICS Microbial Community DNA Standard II

Libraries were prepared using a single protocol from (A) 10 ng and 100 ng of the ZymoBIOMICS Microbial Community DNA Standard(Zymo Research, Catalog #D6306), and (B) 10 ng and 100 ng of the ZymoBIOMICS Microbial Community DNA Standard II (LogDistribution) (Zymo Research, Catalog #D6311). Libraries were pooled and sequenced on an Illumina MiSeq (2 x 75 bases). 1.4
million paired-end reads from each library were sampled (seqtk v1.3), adapter-trimmed (seqprep v0.1) and aligned to a composite
reference genome (bowtie2 v2.4.5). 1,000 bp windows of constituent genomes were counted (bedtools 2.30.0) and compared acrossreplicates and input levels. High correlation was observed between replicates and between inputs.

CONCLUSIONS
Shorter hands-on time
R2 = 0.92

R2 = 0.92R2 = 0.92

NEBNext UltraExpress DNA and UltraExpress FS DNA
library preparation workflows:

R2 = 0.92• Sample in library out in < 2 hours
Fewer steps & consumables

R2 = 0.92

R2 = 0.92

R2 = 0.92

R2 = 0.93

Libraries were prepared using the NEBNext UltraExpress FS DNA protocol from (A) 10 ng and 100 ng of the ZymoBIOMICS®
Microbial Community DNA Standard (Zymo Research®, Catalog #D6306), and (B) 10 ng and 100 ng of the ZymoBIOMICS Microbial
Community DNA Standard II (Log Distribution) (Zymo Research, Catalog #D6311). Libraries were pooled and sequenced on an
Illumina MiSeq® (2 x 75 bases). 750,000 paired-end reads from each library were sampled (seqtk v1.3), adaptor-trimmed (seqprepv0.1) and aligned to a composite reference genome (bowtie2 v2.4.5). 1,000 bp windows of constituent genomes were counted(bedtools 2.30.0) and compared across replicates and input levels. High correlation was observed between replicates and betweeninputs.

Automation friendly volumes

Flexibility with samples types & protocols• Single tube library prep reduces consumables• One protocol fits wide input range and diverse sampletypes• Produce high yields and quality sequencing metrics• Pipetting/reaction volume automation friendlyAuthors would like to acknowledge the technical assistance provided by Dora Posfai, Kristen Augulewicz, and Harry Bell at the New England Biolabs’
Sequencing Core Facility.