Poster: Ambient-Stable Library Preparation Reagents. Next Generation Sequencing(NGS) is widely used to answer clinical questions and drive precision health, but challenges remain for its full democratization. This work introduces a solution to remove cold-chain dependency — a novel and unique set of lyophilized reagents for enzymatic DNA fragmentation and NGS library preparation.
Democratizing Access to NGS for Precision Oncology Through Ambient-Stable Library Preparation Reagents
L. GASIŪNAITĖ, K. Y. CHAN, T. FOUQUEAU, K. KANIUKA, R. M. PORRECA, S. RADIC-HUNTER, M. AMASIO
MERIDIAN LIFE SCIENCE | LONDON, UNITED KINGDOM
INTRODUCTION
LYOPHILIZED DNA LIBRARY PREPARATION
Next Generation Sequencing (NGS) is widely used to answer clinical questions and drive precision health, but challenges remain for its full democratization. Some of the key barriers include translating outputs into actionable results, workflow ease of use, including automation, and the need for cold-chain storage and shipping of reagents, which increases cost and logistical complexity.
PERFORMANCE Library Yield
LYOPHILIZED vs. LIQUID FORMAT COMPARISON
Library Quality Control
Lyophilized DNA library preparation reagents were benchmarked against two competitor kits by comparing final library yield ( Fig. 4 ). Meridian reagents produced higher yields than both market-leading kits, indicating competitive DNA-to-library conversion efficiency.
The impact of reagent lyophilization on library quality was evaluated, focusing on size distribution ( Fig. 2A ) and yield ( Fig. 2B ). Both Bioanalyzer traces and qPCR amplification plots showed matching library size profiles and yields with both lyophilized and liquid reagent formats.
Lyophilized NGS Library Preparation Kit
Quantification of Libraries by qPCR 300000
Figure 4 . Average library yield (pM) of libraries (n = 3), prepared with Meridian lyophilized reagents versus two established competitors, measured by qPCR. Libraries were generated from 100 ng of human gDNA, fragmented using a Covaris ultrasonicator. Lyophilized Meridian library preparation Kit and two established competitor DNA library preparation kits were used. Libraries were constructed using standard protocols from each kit and Illumina adapters. Libraries were quantified using the Meridian qPCR Library Quantification Kit, MDX039.
Lyophilized
Liquid
Lyophilized NGS Enzymatic DNA Fragmentation Kit
A Library Size
B Library Yield
200000
This work introduces a solution to remove cold-chain dependency — a novel and unique set of lyophilized reagents for enzymatic DNA fragmentation and NGS library preparation.
100000
0
Meridian Competitor 1
Competitor 2
Figure 2. ( A ) Electropherograms of libraries, prepared using lyophilized (red) or liquid (blue) reagents; ( B ) Amplification plots for the same libraries quantified by qPCR with P5 and P7 primers. Libraries were generated from 100 ng of gDNA from Escherichia coli MG1455 strain, fragmented using a Covaris ultrasonicator. Libraries were constructed using a standard MDX219 kit workflow with Illumina adapter ligation and PCR amplification (4 cycles). Library size distribution was evaluated on the Agilent 2100 Bioanalyzer system with Agilent High Sensitivity DNA Reagents and yield was quantified using the Meridian qPCR Library Quantification Kit, MDX039.
MDX224 Lyophilized NGS Enzymatic DNA Fragmentation Kit
MDX219 Lyophilized NGS Library Preparation Kit
PERFORMANCE GC Bias
Coverage across extreme-GC regions was benchmarked against an established competitor using libraries prepared from Campylobacter jejuni (30.5% GC) ( Fig. 5A ) and Bordetella pertussis (67.5% GC) ( Fig. 5B ). Meridian library preparation reagents show robust coverage and comparable AT and GC dropout metrics to an established competitor.
LYOPHILIZED DNA FRAGMENTATION The impact of lyophilization on fragmentation reagents was evaluated ( Fig. 1A ), and their performance was compared with a competitor product ( Fig. 1B ). Lyophilized and liquid Meridian reagent formulations produce matched fragmentation profiles and deliver comparable performance to an established commercial alternative.
NGS Metrics
E. coli libraries constructed with either lyophilized or liquid reagents were sequenced to assess the impact of reagent lyophilization on sequencing metrics, including duplication ( Fig. 3A ) and mapping rates ( Fig. 3B ). Comparable sequencing metrics were observed between different reagent formats.
AT Dropout for Campylobacter jejuni A
B
GC Dropout for Bordetella pertussis
0.0 1.0 2.0 3.0 4.0 5.0
0.0 1.0 2.0 3.0 4.0 5.0
A Lyophilized vs. Liquid
B Performance Comparison
3.8
3.9
A Library Duplication Library Lyophilized Reagents
Figure 3 . ( A ) Average duplication and ( B ) mapping rates for WGS libraries (n = 3 per format) prepared with lyophilized or liquid NGS library preparation reagents. Libraries prepared from 100 ng E. coli gDNA were normalized, pooled and sequenced on an Illumina MiSeq using the v2 500-cycle kit (2 × 250 bp). Reads were mapped to the E. coli K12 MG1655 reference genome with BWA-MEM2 and metrics were derived with Picard and samtools.
Lyophilized
Liquid
Meridian
Competitor
1.7
2.0
Avg. Duplication (%)
2.6 ± 0.2 2.7 ± 0.1
Competitor
Competitor
Meridian
Meridian
Liquid Reagents
Figure 5 . GC-bias metrics for libraries prepared with Meridian reagents versus an established competitor (n = 3 per kit). ( A ) AT dropout for reads mapped to Campylobacter jejuni ; ( B ) GC dropout for reads mapped to Bordetella pertussis. gDNA from Campylobacter jejuni and Bordetella pertussis was fragmented on a Covaris ultrasonicator and mixed at an equimolar ratio. 100 ng of the mixed gDNA was used to generate libraries using either the MDX219 workflow or competitor kit workflow with Illumina adapter ligation and PCR amplification (5 cycles). Libraries were normalized, pooled and sequenced on an Illumina NextSeq 2000, P1 600-cycle (2 × 300 bp). Reads were aligned to the respective reference genomes with BWA-MEM2, and metrics were derived with Picard.
B Library Mapping Library
Avg. Mapped Reads (%)
Lyophilized Reagents 99.79 ± 0.04 Liquid Reagents 99.74 ± 0.12
CONCLUSIONS
Figure 1 . The electropherogram and the gel image of the ScreenTape assay of human genomic DNA (gDNA) post-fragmentation with ( A ) Meridian lyophilized (red) vs. liquid (blue) fragmentation reagents; ( B ) Meridian lyophilized reagents (blue) vs. established commercial kit (gold). Human gDNA (1 µ g) was enzymatically fragmented according to the manufacturer’s recommended protocol. Reactions were then purified (1.8x magnetic-bead clean-up) and evaluated on an Agilent TapeStation (D5000).
༎ Lyophilization of reagents for enzymatic DNA fragmentation and NGS library preparation is possible without impact on performance. ༎ The data obtained with lyophilized reagents (fragmentation profile, yield, GC bias) show market-leading performance when compared to key competitor products. ༎ By removing cold-chain requirements, lyophilized reagents broaden access to NGS for clinical applications, including precision oncology.
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