Next-Generation Sequencing (NGS)

Library preparation solutions.

Next-Generation Sequencing (NGS) Library preparation solutions

APPLICATION KEY

Human Diagnostics

Genetic Screening

Next-Generation Sequencing (NGS) has revolutionized genomic research with its throughput, scalability and speed. The applications of NGS in clinical diagnostics are rapidity expanding, particularly in the fields of oncology, microbiology and clinical genetics. In order to be used successfully as a diagnostic tool, data generated by NGS must be reliable and accurate. One of the factors that significantly impacts

the quality of NGS data is the quality and quantity of the library loaded on the sequencer flow cell. A poor-quality library can skew results with uneven coverage or poor yield, affecting the accuracy and reliability of the sequencing. Meridian offers a range of NGS

library preparation solutions compatible with automated workflows for achieving optimal sequencing output.

> Glycerol-Free T4 DNA Ligase

> Glycerol-Free High-Fidelity Pfu

> High-Specificity Pfu HS Mix

> Library clean-up & size selection

> Library Quantification

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Glycerol-Free T4 DNA Ligase

Glycerol-Free T4 DNA Ligase (HC) is a glycerol-free, high concentration enzyme (50 U/μL) that catalyzes the formation of a phosphodiester bond between adjacent 5’ phosphate and 3’ hydroxyl groups in duplex DNA or RNA. It is 2-10x higher in concentration than standard T4 DNA ligases and is glycerol-free and lyophilization compatible. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.

Product Highlights • Efficiently joins blunt and cohesive ends • Repairs single-stranded nicks in duplex DNA, RNA and DNA/ RNA hybrids • Joins double-stranded oligonucleotide linkers or adaptors to DNA • Glycerol-free, suitable for lyophilization • Ideal for use in high-complexity library cloning and NGS library construction

PRODUCT

CAT NO.

VOLUME

20 μL (1,000 Weiss Units) 200 μL (10,000 Weiss Units)

Glycerol-Free T4 DNA Ligase (HC)

MDX200

100U T4 DNA Ligase

No Ligase Control

100U T4 DNA Ligase

No Ligase Control

Free of Detectable of Exo- and Endonuclease Activities

Glycerol-Free T4 DNA Ligase (HC) is highly purified and tested to ensure that it is free of any detectable exonuclease or endonuclease activity that may affect the ligation efficiency of the enzyme. It is also tested to ensure that there is no loss of activity during lyophilization and ambient temperature storage. The very high concentration makes Glycerol-Free T4 DNA Ligase (HC) ideal for using in cassettes for point of care (POC) assays. Glycerol-Free T4 DNA Ligase (HC) is free of detectable exo - and endonuclease activities. Glycerol-Free T4 DNA Ligase (HC) was incubated of for 16 hours at 37 °C with 1 µg of HindIII-digested λ DNA (Lanes 2 and 3) and supercoiled pUC19 DNA (Lanes 5 and 6). The results show no detectable exo- and endonuclease activities.

Glycerol-Free High-Fidelity Pfu (HC)

A high‑concentration (20 U/µL), glycerol‑free, high-fidelity thermostable DNA polymerase, a novel buffer system containing magnesium, dNTPs and excipients, to provide high yield and sensitivity, perfect for target enrichment, NGS library amplification, gene expression, cloning and site-directed mutagenesis.

Product Highlights • High concentration Pfu polymerase allowing for bulk production of PCR master mixes. • Ideal for NGS library amplification • Glycerol-free, ideal for preparation of dried-down PCR tests.

PRODUCT

CAT NO.

VOLUME

REACTION

0.1 mL

500 Rxns

Glycerol-Free High-Fidelity Pfu (HC)

MDX203

10 mL

5,000 Rxns

Multiplex amplification

Glycerol-Free High-Fidelity Pfu (HC), was lyophilized with Lyo-Ready™ Pfu Reaction Buffer, 5x and stored at 37°C for 7 and 28 days. Lyophilization samples were tested in multiplexed PCR amplification of 7 targets (793 bp, 649 bp, 548 bp, 418 bp, 332 bp, 196 bp, 135 bp) against the same enzyme and reaction buffer without lyophilization (Wet Reference) to demonstrate stable after lyophilization allowing for extended storage periods and use in diagnostic tests and POC devices.

High-Specificity Pfu HS Mix

A high-fidelity DNA polymerase with aptamer-based hot-start technology highly suited to target enrichment, NGS library amplification, and cloning applications. High-Specificity Pfu HS Mix provides high yield and sensitivity, as well as superior amplification of GC- and AT-rich targets. The hot-start aptamer binds reversibly to the polymerase which inhibits its activity at ambient temperatures and improves the specificity of the amplification. Product Highlights • Aptamer hot-start for increased specificity • Inhibitor-tolerant (tolerates up to 20% whole blood) PRODUCT CAT NO. VOLUME REACTIONS MDX006 5 mL 200 Rxns

High-Specificity Pfu HS Mix

100 mL 4,000 Rxns

• Robust amplification across different GC-content (29% - 65%) • High-fidelity reduces errors for NGS library amplification • Convenient room temperature reaction set-up • Suitable for multiplexing

Low GC Bias at Both High (over 65%) and Low (under 25%) GC Content

GC bias plot from whole-genome sequencing of a mixture of S. aureus and R. spheroides genomic DNA using High-Specificity Pfu HS Polymerase and supplier K. Libraries were prepared from 100 ng (amplified libraries) or 1 μg (PCR-free library) of genomic DNA from S. aureus and R. spheroides , mixed equimolarly. Library amplification consisted of 10 PCR cycles using High-Specificity Pfu HS Mix (MDX006) or supplier K mix, following the manufacturer’s recommendations. GC bias plots were generated, with %GC content of 100 bp windows on the X axis. Normalized coverage is indicated for the High-Specificity Pfu HS Mix (blue line) and supplier K (green line) and can be compared to a PCR-free reference library (black line), which has the horizontal bias distribution closer to 1 (representing unbiased coverage). The results illustrate High-Specificity Pfu HS Polymerase has a lower bias across the entire range of GC content, compared to the PCR-amplified library from supplier K, especially for GC percentages higher than 65%.

Reference Genome

MDX006

2.0

Supplier K

PCR-Free

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

GC% of 100 base windows

Reference Genome

MDX006

Kapa

PCR-Free

Efficient amplification of DNA containing a wide range of GC-content

M 29% 32% 44% 55% 62% 65%

The following regions from human genomic DNA were amplified: RBB8 161 bp (29% GC), RBB8 450 bp (32% GC), IL 10 417 bp (44% GC), Mpex 418 bp (55% GC), EGFR 525 bp (62% GC), EGFR 380 bp (65% GC). Efficient amplification demonstrated across the different GC content tested the suitability of High-Specificity Pfu HS Mix for applications requiring low GC-bias.

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Library clean-up & size selection

Efficient removal of primers and adapters

Paramagnetic SPRI beads designed for clean-up and size selection of DNA fragments or NGS libraries. Paramagnetic beads selectively bind DNA fragments based on the volume ratio of bead suspension and sample. They are capable of delivering highly purified DNA fragments, efficiently removing contaminants, such as nucleotides, primers, adapters, enzymes, buffer additives and salts.

Electropherogram showing the library size profile after removal of unincorporated adapters and PCR primers (arrows) after using NGS Clean and Selection Beads.

PRODUCT

CAT NO.

VOLUME

50 mL

NGS Clean and Select Beads

MDX041

500 mL

Product Highlights • Highly-reproducible results • Tuneable size selection protocols for single or double sided selection • Compatible with manual and automated workflows

Size distribution after double-sided size selection

For double-sided size selection, supernatant from a right-side selection step (which excludes the largest library fragments) is re-purified using a different ratio of beads, according to the left-sided size selection protocol to exclude the smallest library fragments.

qPCR-based Quantification of NGS Libraries

Library Quantification

qPCR-based assay for the exclusive quantification of adapter-ligated molecules providing accurate quantification of Illumina NGS libraries. Contains pre-diluted standards to minimize pipetting errors, a P5 / P7 Illumina ® specific primer mix and an optimized buffer for dilution of NGS library samples.

PRODUCT

CAT NO.

REACTIONS

Library Quantification Kit

MDX039

500 Rxns / Kit

Product Highlights • Suitable for quantification of all Illumina compatible libraries • Accurate results in less than 90 minutes • Contains a series of six pre-diluted DNA standards for rapid and reliable standard curve generation, providing precise and accurate quantification • 18 sets of standards can be used for 62 libraries on six 96-well plates or 76 libraries on two 384-well plates

Standard curve generated from the amplifications of the 6 pre-diluted DNA standards (blue, 10 pM to 100 aM) and 3 dilutions of an Illumina ® NGS library (red, ten-fold series).

Ordering information: USA 5171 Wilfong Road Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223

Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.meridianbioscience.com/lifescience

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