Ovation® Rapid Library Systems

33 Pages·2016·0.33 MB·English

USER GUIDE
Ovation® Rapid Library Systems
PART NOS. 0319, 0320
Patents, Licensing and Trademarks
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Warranty
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Table of Contents
Contents
I. Introduction .........................................................................................................1
A. Background .......................................................................................................1
B. Performance Specifications ...............................................................................2
C. Library Quantitation ..........................................................................................3
D. Quality Control .................................................................................................3
E. Storage and Stability .........................................................................................3
F. Safety Data Sheet (SDS) ....................................................................................3
II. Components ........................................................................................................4
A. Reagents Provided ............................................................................................4
B. Additional Equipment, Reagents and Labware ................................................5
III. Planning the Experiment .....................................................................................7
A. Input DNA Requirements ..................................................................................7
B. Using Ovation Rapid Multiplex Systems on Illumina NGS Systems .................7
C. Library Quantitation ..........................................................................................8
D. Final Library Storage .........................................................................................8
E. Data Analysis and Parsing Multiplex Libraries ..................................................8
IV. Protocol ...............................................................................................................9
A. Overview ...........................................................................................................9
B. Protocol Notes ..................................................................................................9
C. Agencourt® Purification Beads ........................................................................10
D. Programming the Thermal Cycler ...................................................................11
E. DNA Fragmentation ........................................................................................12
V. Ovation Rapid DR Multiplex Systems 1–8 and 9–16 Protocol ...........................15
A. Setting Up a DR Multiplex Experiment ...........................................................15
B. End Repair .......................................................................................................15
C. Ligation ...........................................................................................................16
D. Final Repair .....................................................................................................17
E. Library Purification ..........................................................................................18
VI. Quantitative and Qualitative Assessment of the Library ....................................20
A. Quantitation Using KAPA Biosystems Products ..............................................20
B. Gel Analysis of KAPA qPCR Product ...............................................................20
VII. Technical Support ..............................................................................................22
VIII. Appendix ...........................................................................................................23
A. Preparation of Ovation Rapid Library Systems Libraries for Cluster
Generation on Illumina Systems .....................................................................23
B. Barcode Sequences for Ovation Rapid Multiplex System Reactions ..............26
C. Frequently Asked Questions (FAQs) ...............................................................27
D. Update History ................................................................................................29
I. Introduction
A. Background
The Ovation® Rapid Library Systems provide a simple, fast and scalable solution for
producing libraries used in next-generation sequencing. These systems enable library
preparation starting with as little as 100 ng of double-stranded DNA without PCR
amplification. The library construction workflow is suitable for a wide range of sequenc-
ing applications including RNA-Seq, Digital Gene Expression (DGE), genomic DNA
sequencing, amplicon sequencing, ChIP-Seq and more.
As shown in Figure 1, the streamlined workflow consists of four main steps: fragmenta-
tion of either genomic DNA or double-stranded cDNA, end repair to generate blunt
ends, adaptor ligation, and final repair to produce the final library. The entire workflow
requires only one bead purification step and no gel purification. Starting with as little as
100 ng of fragmented double-stranded DNA (dsDNA), the protocol can be completed
in fewer than 2 hours and yields libraries ready for cluster formation and either single
read or paired-end sequencing.
In addition to genomic and other double-stranded DNA sources, the Ovation Rapid
Library Systems have been designed for seamless integration with NuGEN’s Ovation
products for cDNA generation, such as Ovation RNA-Seq System V2 (Part No. 7102), to
enable a complete end-to-end solution for transcriptome library construction starting
with total RNA. Importantly, for DNA sequencing applications, low abundance samples
(100 ng or higher) can be input directly to the library construction workflow without the
need for pre-amplification. The absence of PCR steps in the library construction work-
flow makes this library construction method particularly well suited to either high- or
low-GC genomes, which are often susceptible to PCR artifacts.
The Ovation Rapid DR Multiplex Systems 1–8 (Part No. 0319) and 9–16 (Part No. 0320)
each provide eight unique dedicated read adaptors to prepare libraries for multi-
plex sequencing using a dedicated read design strategy with a second sequencing
primer. Either kit may be used for up to 8-plex sequencing or the two kits may be used
together to multiplex up to 16 samples.
1 Ovation Rapid Library Systems
I. Introduction
Figure 1. Ovation Rapid Library System Workflow.
Input DNA
Fragment 5´ 3´
End-repair 5´ P 3´
P
Add adaptors and ligate
Final repair
Bead purification
Cluster formation AATCGGATCGGTAGGAT …
and sequencing TCTCGATGCAAGTGATC …
GTAGCAAAATCCTGAGA …
B. Performance Specifications
The Ovation Rapid Library Systems are designed to produce DNA libraries suitable
for either single read or paired-end sequencing on the Illumina Genome Analyzer IIx/
IIe (GAII), MiSeq, HiScan SQ or HiSeq NGS platforms without gel-based size selection,
using 100–500 ng of double-stranded DNA. When preparing libraries using cDNA
generated from the Ovation RNA-Seq System V2, it is critical to use at least 500 ng –
1 µg of ds-cDNA. The absence of PCR amplification in the workflow eliminates the risk
of unequal amplification or PCR bias introduced as a result of differences in sequence
composition. These systems generate libraries suitable for cluster generation in about
2 hours.
2 Ovation Rapid Library Systems
I. Introduction
C. Library Quantitation
Libraries created using Ovation Rapid Library Systems must be quantified using qPCR.
These libraries cannot be accurately quantified using other means. We recommend
using KAPA Library Quantification kits from KAPA Biosystems for quantitation. See
Section VII, Quantitative and Qualitative Assessment of the Library, for details.
D. Quality Control
Every lot of the Ovation Rapid Library Systems undergoes functional testing to meet
specifications for library generation performance.
E. Storage and Stability
The Ovation Rapid Library Systems are shipped on dry ice and should be unpacked
immediately upon receipt.
Note: This product contains components with multiple storage temperature
requirements.
• Vials labeled Agencourt® Beads (clear cap) should be removed from the top of
the shipping carton upon delivery and stored at 4°C.
• All other components should be stored at –20°C on internal shelves of a freezer
without a defrost cycle.
The kit has been tested to perform to specifications after as many as six freeze/thaw
cycles. Kits handled and stored according to the above guidelines will perform to specifi-
cations for at least six months.
F. Safety Data Sheet (SDS)
If applicable, an SDS for this product is available on the NuGEN website at
www.nugen.com/products/ngs/ovation-rapid-library-systems
3 Ovation Rapid Library Systems
II. Components
A. Reagents Provided
Table 1. Ovation Rapid DR Multiplex System 1–8 and 9–16 Reagents (Part Nos.
0319-32 and 0320-32)
COMPONENT PART NUMBER VIAL LABEL VIAL NUMBER
End Repair Buffer Mix S01532 Blue ER1 ver 4
End Repair Enzyme Mix S01533 Blue ER2 ver 4
End Repair Enhancer S01503 Blue ER3
Ligation Buffer Mix S01534 Yellow L1 ver 4
0319-32 0319-32
S01591 L2DR-BC1
S01592 L2DR-BC2
S01593 L2DR-BC3
S01594 L2DR-BC4
S01595 L2DR-BC5
S01596 L2DR-BC6
S01597 L2DR-BC7
S01598 L2DR-BC8
Ligation Adaptor Mix Yellow
0320-32 0320-32
S01599 L2DR-BC9
S01600 L2DR-BC10
S01601 L2DR-BC11
S01602 L2DR-BC12
S01603 L2DR-BC13
S01604 L2DR-BC14
S01605 L2DR-BC15
S01606 L2DR-BC16
Ligation Enzyme Mix S01535 Yellow L3 ver 4
Final Repair Buffer Mix S01567 Purple FR1
Final Repair Enzyme Mix S01569 Purple FR2
Nuclease-free Water S01001 Green D1
Agencourt Beads S01307 Clear —
4 Ovation Rapid Library Systems
II. Components
B. Additional Equipment, Reagents and Labware
Required Materials
• Equipment
- Covaris S-series Sonication System
- Agilent 2100 Bioanalyzer (optional, for analysis of DNA fragmentation)
- Materials and equipment for electrophoretic analysis of nucleic acids
including 2% agarose gels, DNA ladders (1 kB and 50 bp recommended)
- Real-time PCR System capable of SYBR Green detection
- Microcentrifuge for individual 1.5 mL and 0.5 mL tubes
- 0.5–10 µL pipette, 2–20 µL pipette, 20–200 µL pipette, 200–1000 µL pipette
- Vortexer
- Thermal cycler with 0.2 mL tube heat block, heated lid, and 100 µL
reaction capacity
- Appropriate spectrophotometer and cuvettes, or Nanodrop® UV-Vis
Spectrophotometer for quantitation of fragmented DNA
• Reagents
- Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps
- 1X TE buffer (low EDTA), pH 8.0 (Affymetrix USB products, Cat. #75793) for
input DNA sample
- KAPA Library Quantification kit specified for Illumina and the real-time PCR
system to be used
• Supplies and Labware
- Nuclease-free pipette tips
- 1.5 mL and 0.5 mL RNase-free microcentrifuge tubes
- 0.2 mL individual thin-wall PCR tubes or 8 X 0.2 mL strip PCR tubes or
0.2 mL thin-wall PCR plates
- Magnetic separation device options:
° Agencourt® SPRIPlate Ring Super Magnet Plate (Beckman Coulter,
Cat. #A32782) — for PCR strips, skirted, non-skirted or half-skirted PCR
plates
° Invitrogen™ DynaMag™-96 Side (Invitrogen, Cat. #123-31D) — for PCR
strips, non-skirted or half-skirted PCR plates
° Invitrogen DynaMag-96 Side Skirted (Invitrogen, Cat. #120-27) — for
skirted PCR plates
° Promega MagnaBot® II Magnetic Separation Device (Promega,
Cat. #V8351) — for PCR plates
° Agencourt SPRIStand (Beckman Coulter, Cat. #A29182)
- Disposable gloves
- Kimwipes
- Ice bucket
- Cleaning solutions such as DNA-OFF™ (MP Biomedicals, Cat. #QD0500)
- OPTIONAL: PhiX Control (Illumina, Cat. #FC-110-3001)
5 Ovation Rapid Library Systems
II. Components
To Order
• Affymetrix, Inc., www.affymetrix.com
• Beckman Coulter, www.beckmancoulter.com
• Covaris, www.covarisinc.com
• Illumina, www.illumina.com
• Invitrogen, www.invitrogen.com
• KAPA Biosystems, www.kapabiosystems.com
• MP Biomedicals, www.mpbio.com
• Promega, www.promega.com
• Sigma-Aldrich, Inc., www.sigmaaldrich.com
6 Ovation Rapid Library Systems