NEBNext® Ultra™II FS DNA: A Robust Enzyme-based DNA Library Preparation Method Compatible with Plant and Animal Samples

IntroductionNext generation sequencing (NGS) is currently an important tool used in many fields toanswer biological questions. DNA fragmentation is the critical initial step in the construction ofhigh quality NGS libraries, however, current fragmentation methods create a bottleneck inlibrary preparation throughput. To meet this challenge, we have developed a robust libraryconstruction method (NEBNext Ultra II FS) that integrates enzyme-based DNA fragmentationwith end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube.This method eliminates the need for expensive equipment to fragment DNA; moreover, theoptimized workflow reduces the numerous cleanup and liquid transfer steps, reducing thetime, cost, and errors associated with library construction.

The robustness of the Ultra II FS DNA library preparation workflow was tested using genomicDNA from a variety of sources including the model organism Arabidopsis thaliana, the less-documented genome of Cannabis sativa, and Sus scrofa (pig). Libraries were prepared froma range of DNA inputs to achieve different insert sizes with or without PCR amplification. Alllibraries were sequenced, reads aligned to the appropriate reference genome, and qualitymetrics generated using Picard tools. Compared with the traditional, mechanical shearingbased library preparation method, Ultra II FS is significantly easier to automate, has higherlibrary conversion rate and similar or superior sequencing quality. We further discuss severalapplications of Ultra II FS in plant and animal research, including genome assembly andsample quality control.

Streamlined workflow

Karen R. McKay1, Lynne Apone1, Vaishnavi Panchapakesa1, Pingfang Liu1, Brittany Sexton1, Yvonne Helbert2, Lei Zhang2,Bradley W. Langhorst1, Timur Shtatland, Maggie Heider1, Chen Song1, Christine Sumner1, Christine Rozzi1, Melissa Arn1, Daniela Munafo1,

Fiona Stewart1, Kevin J. McKernan2, Eileen T. Dimalanta1 and Theodore B. Davis1

1New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA. 2Medicinal Genomics Corp., Woburn, MA 01801, USA

Enzyme based fragmentation combined with NEBNext® Ultra™ II DNA Library Preparation for Illumina®

Hands-on time: 8-14min.Total time: 1.5-3hrs

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 by adaptor ligation in the same tube. PCR amplified and PCR-free libraries were sequenced, reads aligned to the appropriate reference genome, and quality metrics generated using Picard tools.

Single tube

IntactDNA ST

EPS

AdaptorLigation

Clean-upSize Select

Amplify Clean-upFragmentEnd RepairdA-Tail

www.NEBNext.com NEW ENGLAND BIOLABS® and NEBNEXT® are registered trademarks and ULTRATM is a trademark of New England Biolabs, Inc. ILLUMINA®, MISEQ® and NEXTSEQ® are registered trademarks of Illumina, Inc. COVARIS® is a trademark of Covaris, Inc. BIOANALYZER® is a registered trademark of Agilent Technologies.

Ultra II FS enables time-dependent fragmentation

A

B

A) 100 ng of input genomic DNA was incubated with the NEBNext Ultra II FS Enzyme Mix and ReactionBuffer for 5, 10, 15, 20, 25, 30 and 40 minutes at 37 ̊C, followed by 65 ̊C for 30 minutes. After clean-upusing NEBNext Sample Purification Beads, size was assessed using the Agilent Bioanalyzer. B) Librarieswere constructed using the NEBNext Ultra II FS kit and 100 ng of input, with fragmentation times of 5, 10,15, 20, 25, 30 and 40 minutes, and 4 PCR cycles. Size selection was not performed. After clean-up usingNEBNext Sample Purification Beads, size was assessed using the Agilent Bioanalyzer. NEBNext Ultra II FSshows expected final library sizes consistent with the fragmentation sizes seen in A). Human NA19240genomic DNA was used to generate the figures above.

Results

Robust fragmentation independent of DNA input and buffer

A

B

A) Libraries were made using 100 ng input genomic DNA using the NEBNext® Ultra™ II FS kit. Libraries werefragmented to generate 200 bp inserts (320 bp libraries) from DNA resuspended in H2O, 10mM Tris, 0.1X TEor 1X TE . Library size distribution was assessed using the Agilent® Bioanalyzer®. B) Libraries were preparedfrom input genomic DNA using the input amounts shown. NEBNext® Ultra™ II FS libraries were preparedusing a 20-min fragmentation time. Library size was assessed using the Agilent® Bioanalyzer®. Low input (1ng and below) libraries were loaded on the Bioanalyzer® without dilution. High input libraries were loaded witha 1:5 dilution in 0.1X TE. Human NA19240 genomic DNA was used to generate the figures above.

Inferred Kit

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of in

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Insert Sizes

MedicinalGenomics_Ch3_size746bp_pl..

Library

MedicinalGenomics_Ch3_size746bp.1

The trend of average of % of inserts for Insert Size broken down by Sample and Inferred Kit. Color shows details about Library. Size shows details about Project.Details are shown for read_group_id. The data is filtered on User, Exclusions (Inferred Input (ng),Inferred Kit,Insert Size,Library,Project,read_group_id,Sample), RunName and Date. The User filter keeps bsexton. The Exclusions (Inferred Input (ng),Inferred Kit,Insert Size,Library,Project,read_group_id,Sample) filter specifies a set.The Run Name filter keeps multiple members. The Date filter includes the last 8 months. The filter associated with this field ranges from 6/1/2018 to 1/31/2019. The viewis filtered on Project, Sample and Library. The Project filter keeps KD_5.15.18_UIIFS_OTClot2_SClot_10XdATPbuffer and MedicinalGenomics_Ch3_size746bp_plant.The Sample filter keeps multiple members. The Library filter keeps multiple members.

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Library, Inferred Amplification

MedicinalGenomics_Ch3_size746bp.1, +PCR

The trends of Avg. Normalized Coverage and % of Total Avg. Num Windows for Pct Gc broken down by Sample. For pane Average of Normalized Coverage: Color shows details about Libraryand Inferred Amplification. Size shows details about Inferred Kit. Details are shown for Avg. Normalized Coverage. The data is filtered on User, Project, Run Name and Date. The User filterkeeps bsexton. The Project filter keeps KD_5.15.18_UIIFS_OTClot2_SClot_10XdATPbuffer and MedicinalGenomics_Ch3_size746bp_plant. The Run Name filter keeps multiple members. TheDate filter includes the last 8 months. The filter associated with this field ranges from 6/1/2018 to 1/31/2019. The view is filtered on Sample and Library. The Sample filter keeps multiplemembers. The Library filter keeps multiple members.Reliable SNP detection with 1ng Arabidopsis DNA

Conclusions

Acknowledgements

A

B

Large insert size libraries can be generated with theNEBNext® Ultra™ II FS kit. 250 ng of Jamaican LionFemale leaf DNA was fragmented for 5 minutes. Afteradaptor ligation, a bead based size selection wasperformed for large fragment size. The library wasamplified for 5 cycles using the NEBNext Ultra II Q5master mix and sequenced on an Illumina MiSeq® (2 X150 bp). Reads were aligned to Jamaican Lionreference genome (August 2018 assembly) usingBowtie 2.2.4.A) Insert size was calculated using Picard Metrics B)GC coverage information was calculated using Picard’sCollectGCBiasMetrics (v1. 117). Expected normalizedcoverage of 1.0 is indicated by the horizontal grey line,the number of 100 bp regions at each GC% is indicatedby the vertical grey bars, and the colored line representsthe normalized coverage for the library.

Large insert size with Cannabis sativa female leaf

Quality control of reagents using Sus scrofa

Large Inserts > 500 bp

NEBNext® Ultra™ II FS

Input DNA (1 ng and 50 ng)

Illumina MiSeq®

(2 X 150 bp)

SNP detection

FreeBayes

Sus Scrofa 88%

Mammalia

Eukaryota

Root

11% No hits0.004% Bacteria0.001% Viruses0.02% other Root

100ng Input DNA

NEBNext® Ultra™ II FSPCR Free

Detect contamination(Human, E.coli)

This germ line variant summary plot shows that the SNPs are represented comparably regardless of input amount, demonstrating that NEBNext Ultra II FS can be used for accurate SNP detection with minimal sample input.

An uncommon source of DNA was used as a quality control measure for detecting contaminants such as human and bacterial DNA in our final libraries, which indicates the purity of our library preparation reagents.

Authors would like to acknowledge the technical assistance provided by Laurie Mazzola, Danielle Fuchs, and Aine Quimby at the New England Biolabs’ Sequencing Core Facility.

Insert Size (MiSeq)

GC Bias

§ Ultra™ II FS enables high-quality library construction from a broad range of DNA quantities and qualities and is compatible with plant and animal samples.

§ Ultra™ II FS maintains reliable SNP detection with minimal sample input.

§ Large insert sizes can be generated with Ultra™ II FS.

§ Ultra™ II FS provides a robust, time-dependent fragmentation amenable for manual or automated library preparation.

0.975% Other Mammalia