Next-Generation Sequencing (NGS) Library preparation solutions
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
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) • 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
High-Specificity Pfu HS Mix
High-Specificity Pfu HS Mix continued
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. 1.8 2.0
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. End-repair and ligation steps were carried out using Meridian NGS ER Enzyme Mix (MDX040) and NGS Ligase (MDX037) for all the samples. 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
GC% of 100 base windows
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%.
High-Specificity Pfu HS Mix
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.
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
Supplier 2 Supplier 3
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.
Enzymes and buffers necessary for end repair, A-tailing, ligation and PCR amplification. Reactions can be performed in the same tube as reaction buffers have been optimized to work together to provide maximum reaction efficiency and conversion rates, while eliminating clean-up steps and minimizing sample loss. High-Fidelity Pfu and its buffer have been developed to ensure even coverage of the results with minimal error rate incorporation. Enzymes for PCR-amplified and PCR-free Libraries
NGS Enzymes NGS ER Enzyme Mix A mix that processes both 3’ and 5’ overhangs generating products that are 5’ phosphorylated with 3’ A-overhang NGS Ligase Enzyme that catalyzes the ligation of adapters to end-repaired DNA fragments High-Fidelity Pfu DNA polymerase with 3’-5’ proofreading exonuclease activity, with an error rate of 3.0 x 10 -6 generating blunt-ended amplicons up to 5 kb in length
Buffers NGS End-Repair Buffer, 5x Optimized for use with NGS ER Enzyme Mix (Cat# MDX040 ) NGS Ligase Buffer, 5x Optimized for use with NGS Ligase (Cat# MDX037 ) NGS High-Fidelity Pfu Buffer, 10x Optimized for use with High-Fidelity Pfu (Cat# MDX003 )
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.
50 mL 500 mL
NGS Clean and Select Beads
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.
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.
Library Quantification Kit
500 Rxn / 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).
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