Library preparation solutions.
Enzymes for Next Generation Sequencing (NGS) Democratizing NGS sequencing through innovative reagents
With the reduction in cost and increased portability of sequencing instruments, consumables, and data analysis tools, NGS is becoming more accessible, affordable, and user-friendly for a wider range of researchers, clinicians, and industries. Recent technological advances have opened the door to smaller and more compact sequencing instruments that have a reduced footprint, user-friendly interface, simplified workflow, and lower sample requirements. These advantages reduce the cost of performing NGS and increase its portability, enabling researchers to conduct sequencing experiments in various settings, including fieldwork and point-of-care diagnostics. However, a continuous challenge for NGS researchers has been the requirement of cold-chain shipping and storage for assay reagents. Ensuring the integrity of the cold chain throughout the supply chain logistics, especially in environments with unreliable electricity or limited refrigeration capacity, has resulted in high costs and logistical hurdles, hindering the global accessibility of NGS technology. Meridian’s glycerol-free NGS enzyme format enables lyophilization, providing a sustainable alternative to reduce carbon emissions associated with cold-chain logistics. This eradicates reliance on energy-intensive refrigeration and freezing methods. Additionally, eliminating cold-chain requirements simplifies transportation logistics, reducing packaging materials and transportation costs. In addition to the shipping and storage stability enabled by being glycerol-free, the enzymes are available in a high-concentration format, meeting the demands for miniaturization, often required in point-of-care devices. This format not only allows for the utilization of smaller reagent volumes but also maximizes the sample input quantity while ensuring consistent performance. Meridian’s new glycerol-free high-concentration enzymes empower NGS assay developers to engineer the next generation of NGS diagnostic tests and workflows.
Meridian’s NGS Library Prep Solutions
MDX207
MDX170
Glycerol-Free T4 DNA Polymerase (HC)
Glycerol-Free Taq HS High Conc. DNA Polymerase
MDX208
MDX203
Glycerol-Free DNA Pol I Klenow Fragment (HC)
Glycerol-Free High-Fidelity Pfu (HC)
MDX206
MDX041
Glycerol-Free T4 Polynucleotide Kinase (HC)
NGS Clean and Select Beads
MDX200
MDX039
Glycerol-Free T4 DNA Ligase (HC)
NGS Library Quantification
Key product features
G lycerol-free enzymes are fully active after lyophilization and storage at ambient temperature for a minimum of 12 months
H igh-concentration format is ideal for assays requiring reduced volumes, such as miniaturized point-of-care devices
High-concentration format also allows for flexibility and maximization of the sample input quantity to increase assay sensitivity
Includes all enzymes required for development of NGS library prep kit in a glycerol-free format
www.meridianbioscience.com/lifescience
1
Meridian’s Glycerol-Free NGS Library Preparation Solution Overview of library preparation by fragmentation and relevant Meridian products to perform each step. Fragments with 5’ and 3’ overhangs are repaired to create blunt ends, these are then phosphorylated and A-tailed, leaving ends amenable to adaptor ligation. Sequencing adaptors are then ligated to create complete sequencing-ready library fragments.
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
STEP 1 Fragmentation Physical or enzymatic
5’
5’
3’
3’
5’ 3’
5’ 3’
3’
5’
5’
3’
5’
5’
5’
5’
3’
3’
5’ overhang 3’ 3’
3’ overhang
5’ 3’ 5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
3’
3’
5’
5’
5’
5’
3’
3’
5’ overhang
5’ overhang
3’ overhang
3’ overhang
5’
5’
3’
3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
3’
5’ 3’ 5’ 3’ 5’ 3’ 5’
5’
3’
5’ 5’ 5’ 3’
5’ 5’ 5’ 3’
3’ 3’
3’ 3’
5’ overhang
3’ overhang
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
STEP 2
Create blunt ends
(3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction)
3’ 3’
5’ 5’
5’ 5’
3’ 3’
5’ 5’ overhang 5’ overhang 5’ overhang P 3’
3’ overhang 3’ overhang 3’ overhang 5’ 3’ 5’ 3’
Glycerol-Free T4 DNA Polymerase (HC) (MDX207) SEE PAGE 3 Glycerol-Free DNA Pol I Klenow Fragment (HC) (MDX208) SEE PAGE 3
5’
3’ 5’ 3’
(3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction)
5’ 3’
5’ 3’
5’
5’
3’
3’
5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
(3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction)
P
P
5’
5’
3’
5’
P
3’
3’
(3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity) 5’ 3’ 3’ 5’ P 5’ P
P 5’ (Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction) 5’ 3’
5’ 3’
5’ 3’
3’
3’
P
5’
STEP 3
Phosphorylation
A
Glycerol-Free T4 Polynucleotide Kinase (HC) (MDX206) SEE PAGE 4
3’
P P
5’ 5’
3’
3’
5’
P 3’
P 5’ 5’
3’ 3’
A
P A
A
5’
3’
3’ 3’ P 5’ P 5’ P 5’ A- 5’ A 3’ 3’ A 3’ -T A -T
P 3’
-A 5’ 5’ 5’ 5’ 5’ A A
P P 3’ P
3’ 5’
P
A
-T
5’
3’ 3’ P
STEP 4
A-tailing
A-
T- A 5’
P
Glycerol-Free Taq HS 50 U/ µ L (MDX170) SEE PAGE 5
Adaptor -A T- -A T-
3’ 3’
Adaptor
A A P 5’ A- 3’ A -T A-
P P
3’
Adaptor
Adaptor
Adaptor
Adaptor
-A
T-
5’
P
Adaptor
-T -T A-
Adaptor -A
Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor
Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor
STEP 5
Adaptor ligation
A- A-
T- T-
-A
Glycerol-Free T4 DNA Ligase (HC) (MDX200) SEE PAGE 5
-T
-A
T-
Glycerol-Free High-Fidelity Pfu (HC) (MDX203) SEE PAGE 6 STEP 6 Library amplification
2
STEP 2 CREATE BLUNT ENDS
Glycerol-Free T4 DNA Polymerase (HC)
Cat #MDX207
VOLUME REACTIONS 50 μ L 500 Rxns 500 μ L 5,000 Rxns
Glycerol-Free T4 DNA Polymerase (HC) (MDX207) is supplied at concentration 20 U/ μ L and is a template dependent DNA polymerase that catalyzes the addition of nucleotides to the 3’ end of a DNA strand, extending it in the 5’ to 3’ direction. It also possesses 3’ to 5’ exonuclease activity, allowing it to remove nucleotides from the 3’ end of DNA strands but lacks a 5´to 3´ exonuclease activity. It can be used for generating blunt ends on any duplex DNA molecule and for labelling DNA by replacement synthesis. In NGS library preparation it can be used separately or as part of an End-Repair Mix, where it can blunt DNA ends of double-stranded DNA fragments for subsequent ligation of adaptors.
Product Highlights • Blunting of DNA ends by “filling in” or “polishing” the ends of double-stranded DNA fragments, for cloning or generating NGS libraries. • Sanger sequencing, to remove fluorescent dye-labeled nucleotides as they are incorporated into the growing DNA strand. • Concentration 20 U/ µ L
No loss of activity after lyophilization
(Right) Glycerol-Free T4 DNA polymerase (HC) (MDX207) was lyophilized in its reaction buffer, and the stability was tested in an accelerated stability study. The lyophilized MDX207 ( red ) was incubated at 37°C for 1 month and its DNA polymerization activity was tested against a non-lyophilized aliquot stored at -20°C ( blue ). Results suggest that the lyophilized MDX207 is active following accelerated stability tests with projected 12 months stability at ambient temperature.
Liquid | Lyophilized
Glycerol-Free DNA Pol I Klenow Fragment (HC)
Cat #MDX208
VOLUME REACTIONS 200 μ L 10,000 Rxns 1 mL 50,000 Rxns
Glycerol-Free DNA Pol I Klenow Fragment (HC) (MDX208) is supplied at 50 U/ μ L and is a large N-terminal truncated protein fragment produced from E. coli DNA polymerase I, which retains polymerase and 3’-5’ exonuclease activity but has lost 5’-3’ exonuclease activity. It can be used to fill in 5´-protruding ends and is ideal for DNA blunting by 3’ overhang removal and fill-in of 5’ overhangs prior to adapter ligation in next-generation sequencing library preparation.
Product Highlights • DNA blunting for NGS adapter ligation. • Labeling by fill-in of 5’-overhangs of dsDNA. • Concentration 50 U/ µ L
No loss of activity after lyophilization
Fast kinetics with high performance
Meridian | NEB | Thermo | Qiagen
(Above) Glycerol-Free DNA Pol I Klenow Fragment (HC) was used in a primer extension assay. Glycerol-Free DNA Pol I Klenow Fragment (HC) was used at 5 U final in reaction with 4 ng of template and Lyo-Ready Klenow Reaction Buffer and compared to other suppliers under the same conditions. The results demonstrate that Glycerol-Free DNA Pol I Klenow Fragment (HC) has a faster reaction rate and higher end fluorescence than most suppliers.
Liquid | Lyophilized
www.meridianbioscience.com/lifescience (Above) Accelerated stability studies of Glycerol-Free DNA Pol I Klenow Fragment (HC) at 37°C in liquid ( blue ) or lyophilized ( red ) format. The results demonstrate that Glycerol-Free DNA Pol I Klenow Fragment (HC) is active following accelerated stability tests with at least a projected 12 months stability at ambient temperature.
3
STEP 3 PHOSPHORYLATION
Glycerol-Free T4 Polynucleotide Kinase (HC)
Cat #MDX206 VOLUME REACTIONS 25 μ L 500 Rxns 250 μ L 5,000 Rxns
Glycerol-Free T4 Polynucleotide Kinase (HC) (MDX206) is supplied at 20 U/ μ L and is used to phosphorylate the 5’ end of both DNA and RNA for subsequent ligation by catalyzing the transfer of the γ -phosphate from ATP to the 5´ hydroxyl of single stranded and double stranded DNA. Glycerol-Free T4 Polynucleotide Kinase (HC) is used in NGS library preparation, either separately or as part of an End-Repair Mix, where phosphates are added to the 5’-hydroxyl terminus of double stranded DNA for subsequent ligation of adaptors by T4 DNA Ligase.
Product Highlights • 5’ phosphorylation for
subsequent ligation for NGS. • Removes 3’ phosphoryl groups from DNA and RNA. • Concentration 20 U/ µ L
Fast kinetics with high performance
A
B
0.012
0.01
0.008
0.006
0.004
0.002
0
NEB
Meridian
NEB | MDX206
(Above) Increase in fluorescence over time and B/ phosphorylation reaction rate of Glycerol-Free T4 Polynucleotide Kinase (HC) ( blue ) and NEB’s T4 Polynucleotide Kinase ( red ) at a concentration of 0.005 U/ μ L. The results demonstrate that the glycerol-free enzymatic activity of T4 Polynucleotide Kinase produced by Meridian is comparable to glycerol-containing enzymes from other suppliers.
No loss of activity after lyophilization
(Left) Accelerated stability studies of Glycerol-Free T4 Polynucleotide Kinase at 37°C for two months in lyophilized ( red ) compared to the liquid ( blue ) format, stored at -20°C. The results demonstrate that Glycerol-Free T4 Polynucleotide Kinase is not affected by lyophilization and subsequent storage at ambient temperature fully retaining its activity when rehydrated.
Liquid | Lyophilized
4
STEP 4 A-TAILING
Glycerol-Free Taq HS High Conc. DNA Polymerase
Cat #MDX170
VOLUME
UNITS
No loss of activity after lyophilization (Right) Glycerol-Free Taq HS High Conc. DNA Polymerase, was lyophilized with its reaction buffer and stored at 37°C for 1 month Lyophilized enzyme ( red ) was tested in PCR amplification and compared with the enzyme stored at -20°C, not lyophilized ( blue ). Results demonstrate that the Glycerol-free Taq HS is not affected by lyophilization and subsequent storage at ambient temperature, fully retaining its activity when rehydrated. generating 3’-dA-tailed DNA fragments as it has terminal transferase activity and naturally leaves a 3’ terminal adenine making it ideal for ‘sticky end’ ligation of sequencing adapters during NGS library preparation. Glycerol-Free Taq HS High Conc. DNA Polymerase (MDX170) is supplied as a blend of high‑performance glycerol‑free Taq DNA Polymerase with a Taq hot-start antibody. Glycerol-Free Taq HS High Conc. DNA Polymerase can be used for
Product Highlights • H igh concentration polymerase allowing for reduced volume in a miniaturized format. • C an be used for addition of sequencing adapters during NGS library preparation. • Concentration 50 U/ µ L 20 μ L
1,000 500 μ L 25,000
Liquid | Lyophilized
STEP 5 ADAPTOR LIGATION
Glycerol-Free T4 DNA Ligase (HC) (MDX200) is a high concentration enzyme (50U/ μ L) that catalyzes the formation of a phosphodiester bond between adjacent 5’ phosphate and 3’ hydroxyl groups in duplex DNA. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex DNA. Glycerol-Free T4 DNA Ligase (HC)
Cat #MDX200
VOLUME
UNITS
Product Highlights • Efficiently joins blunt and cohesive ends. • Repairs single-stranded nicks in duplex DNA, RNA and DNA/RNA hybrids.
1,000 Weiss Units 10,000 Weiss Units
20 μ L
200 μ L
Free of detectable of exo- and endonuclease activities
• Joins double-stranded oligonucleotide linkers or adaptors to DNA, for use in NGS library construction and cloning. • Concentration 50 U/ µ L
Hindlll-digested λ DNA
Supercoiled pUC19 DNA
100 U T4 DNA Ligase
100 U T4 DNA Ligase
No Ligase Control
100 U T4 DNA Ligase
100 U T4 DNA Ligase
No Ligase Control
(Left) 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 µ g 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 that may affect the ligation efficiency of the enzyme.
www.meridianbioscience.com/lifescience
5
STEP 6 LIBRARY AMPLIFICATION
Glycerol-Free High-Fidelity Pfu (HC) (MDX203) is a high-fidelity, thermostable DNA-dependent DNA polymerase from Pyrococcus furiosus . The 3’ to 5’ proofreading exonuclease activity of Pfu polymerase makes it suitable for applications that require high-fidelity DNA synthesis or blunt-ended PCR fragments. Glycerol-Free High-Fidelity Pfu (HC) is supplied in a glycerol-free storage buffer and is accompanied by a 5x Reaction Buffer that contains magnesium, dNTPs and excipients required for lyophilization. Glycerol-Free High-Fidelity Pfu (HC)
Cat #MDX203 VOLUME
UNITS
Product Highlights • High concentration Pfu
100 μ L 500 10 mL 20,000
polymerase allowing for bulk production of PCR master mixes. • Ideal for NGS library amplification. • Glycerol-free, ideal for preparation of dried-down PCR tests • Concentration 20 U/ µ L
Maintains multiplexing performance after lyophilization
(Left) Glycerol-Free High-Fidelity Pfu (HC) (MDX203), stored at 37°C for 1 and 4 weeks. Lyophilized samples were tested against the same enzyme and reaction buffer without lyophilisation (Wet Reference) in multiplexed PCR amplification of 7 targets of various GC content: 793 bp (32% GC), 649 bp (54% GC), 548 bp (60% GC) , 418 bp (48% GC), 332 bp (48% GC), 196 bp (51% GC), 135 bp (55% GC). Results show that the lyophilized MDX203 is stable with no loss of activity for at least 1 month at 37°C with projected ambient temperature stability of 1 year.
Wet Reference
Lyo Day 7 at 37°C
Lyo Day 28 at 37°C
Lyo Day 0
6
ADDITIONAL NGS PRODUCTS
Meridian’s NGS Clean and Select Beads (MDX041) are 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 delivering highly purified DNA fragments, efficiently removing contaminants, such as nucleotides, primers, adapters, enzymes, buffer additives and salts. NGS Clean and Select Beads
Cat #MDX041
VOLUME 50 mL 500 mL
Product Highlights • Paramagnetic beads for
clean-up and size selection. • Tuneable size selection protocols for single or double-sided selection. • C ompatible with manual and automated workflows.
Left-side and right-side size selection of DNA fragments
(Left) 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, in a left-side purification step to exclude the smallest library fragments.
Library Quantification Kit
Cat #MDX039
VOLUME REACTIONS 5 mL 500
Meridian’s Library Quantification Kit (MDX039) is a qPCR-based assay for the exclusive quantification of adapter-ligated molecules providing accurate quantification of Illumina NGS libraries. It 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 Highlights • Suitable for quantification of all Illumina compatible libraries. • Accurate results in less than 90 minutes. • C ontains six pre-diluted DNA standards for rapid and reliable standard curve generation.
qPCR-based Quantification of NGS Libraries
(Left) 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).
6 pre-diluted DNA standards | Illumina ®
www.meridianbioscience.com/lifescience
7
Ordering Information
Glycerol-Free NGS Enzymes
MDX207
Glycerol-Free T4 DNA Polymerase (HC)
MDX208
Glycerol-Free DNA Pol I Klenow Fragment (HC)
MDX206
Glycerol-Free T4 Polynucleotide Kinase (HC)
MDX170
Glycerol-Free Taq HS High Conc. DNA Polymerase
MDX200
Glycerol-Free T4 DNA Ligase (HC)
MDX203
Glycerol-Free High-Fidelity Pfu (HC)
Supporting NGS Products
MDX041
NGS Clean and Select Beads
MDX039
NGS Library Quantification
MDX003
High-Fidelity Pfu
MDX006
High-Specificity Pfu HS Mix
MDX204
Lyo-Ready T4 Ligase Buffer, 5x
MDX200
RNase Inhibitor (Glycerol Free)
MDX117
55C MMLV-RT
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
Connect with us:
ISO 13485 Certified
05/24
Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8Powered by FlippingBook