Next-Generation Sequencing (NGS)

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.

> High-Specificity Pfu HS Mix

> Library clean-up & size selection

> Library Quantification

www.MeridianLifeScience.com

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 100 mL 4,000 Rxns

High-Specificity Pfu HS Mix

• 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 2.0

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%.

1.8

Reference Genome

MDX006

Supplier K

PCR-Free

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

15

25

35

45

55

65

75

85

GC% of 100 base windows

Reference Genome

MDX006

Kapa

PCR-Free

High sensitivity and yield PCR amplification of a 1.2 kb fragment of the F8 gene, from a five-fold serial dilution of human genomic DNA. All reactions were set up according to the manufacturers’ recommendations. Marker is HyperLadder ™ 50 bp, (BIO-33054) . Meridian’s High-Specificity Pfu HS Mix has higher sensitivity and yield over suppliers.

High-Specificity Pfu HS Mix

Pfu Turbo

Platinum SuperFi

Phusion II

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). Marker is HyperLadder ™ 50 bp, (BIO-33054 ). Efficient amplification was possible across the different GC content tested demonstrating the suitability of High-Specificity Pfu HS Mix for applications requiring low GC-bias.

Suitable for multiplex reactions from inhibitor-rich samples

High-Specificity Pfu HS

Platinum SuperFi

Pfu Turbo Phusion II

Multiplexed PCR amplification of 6 targets (961 bp, 892 bp, 793 bp, 548 bp, 332 bp, 196 bp) directly amplified from 20% whole blood and run in duplicate. Marker is HyperLadder ™ 50 bp, (BIO-33054) . High-specificity Pfu HS Mix is able to outperform other suppliers in the presence of 20% whole EDTA blood in a multiplexed PCR reaction.

www.MeridianLifeScience.com

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 500 mL

NGS Clean and Select Beads

MDX041

Size distribution after double-sided size selection

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

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.

Library Quantification

qPCR-based Quantification of NGS Libraries

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

Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.MeridianLifeScience.com

Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223

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02/22

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