Ovation® Rapid DR Multiplex System 1–96

20 Pages·2016·0.33 MB·English

USER GUIDE
Ovation® Rapid DR Multiplex System 1–96
PART NO. 0328
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Table of Contents
Contents
I. Introduction .........................................................................................................1
A. Background .......................................................................................................1
B. Performance Specifications ...............................................................................2
C. Library Quantification ........................................................................................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 ................................................4
III. Planning the Experiment .....................................................................................6
A. Input DNA Requirements ..................................................................................6
B. Using the Ovation Rapid DR Multiplex System 1–96 on
Illumina NGS Platforms ....................................................................................6
C. Working with the NGS DR Adaptor Plate 1–96 ................................................7
D. Library Quantification ........................................................................................7
E. Amplified Library Storage .................................................................................7
F. Data Analysis and Parsing Multiplex Libraries ..................................................7
IV. Protocol ...............................................................................................................8
A. Overview ...........................................................................................................8
B. Programming the Thermal Cycler .....................................................................8
C. Constructing the Automation Script .................................................................9
V. Quantitative and Qualitative Assessment of the Library ...................................12
A. Quantitative and Qualitative Assessment of the Library .................................12
B. Gel Analysis of KAPA qPCR Product ...............................................................12
VI. Technical Support ..............................................................................................14
VII. Appendix ...........................................................................................................15
A. Barcode Sequences for Ovation Rapid DR Multiplex System 1–96 Reactions 15
B. Using the Ovation Rapid DR Multiplex System 1–96 as a Manual Kit ............15
C. Frequently Asked Questions (FAQs) ...............................................................15
D. Update History ................................................................................................17
I. Introduction
A. Background
The Ovation® Rapid DR Multiplex System 1–96 provides a simple, fast and scalable
solution for producing libraries used in next-generation sequencing. This system
enables library preparation starting with as little as 100 ng of double-stranded DNA
without PCR amplification. The library construction workflow is compatible with a wide
range of sequencing applications including RNA-Seq, Digital Gene Expression (DGE),
genomic DNA sequencing, amplicon sequencing and more. These libraries are suitable
for sequencing on the Illumina Genome Analyzer IIx/IIe (GAII), HiScan™ SQ, MiSeq™
and HiSeq™ 2000/2500 Systems.
While the Ovation Rapid DR Multiplex System 1–96 is primarily intended for use with a
liquid handling robotics system, such as the Caliper Sciclone NGS workstation, it may
also be used for manual library production. For manual processing, refer to Appendix
B: Using the Ovation Rapid DR Multiplex System 1–96 as a Manual Kit.
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 generally
be completed in fewer than two hours and yields libraries ready for quantitation and
cluster formation and either single read or paired-end sequencing. For large numbers
of samples, DNA fragmentation is expected to require additional time, possibly making
the entire library production process longer than two hours.
The Ovation Rapid DR Multiplex System 1–96 has been designed for seamless integra-
tion with NuGEN’s Ovation® RNA-Seq System V2 and Ovation RNA-Seq FFPE System
(Part Nos. 7102 and 7150) to enable a complete end-to-end solution for transcriptome
library construction starting with total RNA.
The Ovation Rapid DR Multiplex System 1–96 (Part No. 0328) contains reagents for
production of up to 96 barcoded libraries. Each kit provides 96 unique 8-base dedi-
cated read (DR) barcoded adaptors to prepare libraries for multiplex sequencing using
a dedicated read design strategy with a second sequencing primer.
1 Ovation Rapid DR Multiplex System 1–96
I. Introduction
Figure 1. Ovation Rapid DR Multiplex System 1–96 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 DR Multiplex System 1–96 is designed to produce DNA librar-
ies suitable for either single read or paired-end sequencing on the Illumina Genome
Analyzer IIx/IIe (GAII), MiSeq, HiScan SQ or HiSeq 2000/2500 systems without gel-
based size selection, using 100–500 ng input of double-stranded DNA. The absence of
PCR amplification in the workflow eliminates the risk of unequal amplification or PCR
bias introduced as a result of differences in template composition.
When preparing libraries When preparing libraries using cDNA, it is critical to use at least 0.5–1 µg of ds-cDNA
using cDNA, it is critical prepared using the Ovation RNA-Seq System V2 (Part No. 7102) or Ovation RNA-Seq
to use at least 0.5–1 µg of
FFPE System (Part No. 7150).
ds-cDNA prepared using
the Ovation RNA-Seq
System V2 (Part No. 7102)
or Ovation RNA-Seq FFPE
System (Part No. 7150).
2 Ovation Rapid DR Multiplex System 1–96
I. Introduction
C. Library Quantification
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 quantification. See
Section V. Quantitative and Qualitative Assessment of the Library for details.
D. Quality Control
Every lot of the Ovation Rapid DR Multiplex System 1–96 undergoes functional testing
to meet specifications for library generation performance.
E. Storage and Stability
The Ovation Rapid DR Multiplex System 1–96 is shipped on dry ice and should be
unpacked immediately upon receipt.
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
specifications 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 DR Multiplex System 1–96
II. Components
A. Reagents Provided
Table 1. Ovation Rapid DR Multiplex System 1–96 Reagents (Part No. 0328-96)
0328 VIAL VIAL
COMPONENT MRV*
PART NUMBER LABEL NUMBER
End Repair Buffer Mix S01686 Blue ER1 ver 3 609 µL
End Repair Enzyme Mix S01687 Blue ER2 ver 4 101 µL
End Repair Enhancer S01688 Blue ER3 275 µL
Ligation Buffer Mix S01689 Yellow L1 ver 4 950 µL
Ligation Enzyme Mix S01690 Yellow L3 ver 4 238 µL
Final Repair Buffer Mix S01695 Purple FR1 2573 µL
Final Repair Enzyme Mix S01696 Purple FR2 135 µL
Nuclease-free Water S01001 Green D1 1220 µL
NGS DR
Ligation Adaptor Plate S01697 — Adaptor 3 µL/well
Plate 1–96
*Minimum Recoverable Volume
Note: SPRI beads (Agencourt® RNAClean® XP) are required for the final purification
step in the Ovation Rapid DR Multiplex System 1–96 library process. This product must
be obtained from Beckman Coulter. NuGEN does not supply SPRI beads in this kit due
to the variances in purification method implementation on different liquid handling
platforms.
B. Additional Equipment, Reagents and Labware
Required Materials
• Equipment
- Covaris S-series Sonication System
- 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
4 Ovation Rapid DR Multiplex System 1–96
II. Components
- 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 quantification of fragmented DNA
• Reagents
- Agencourt® RNAClean® XP Kit (Beckman Coulter, Cat. #A63987)
- KAPA Library Quantification Kit specified for Illumina sequencing platforms
and for the qPCR platform to be used
- Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps
- 1X TE buffer (low EDTA), pH 8.0 (Affymetrix USB products, Cat. #75793)
• 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. #12331D) — for PCR
strips, non-skirted or half-skirted PCR plates
° Invitrogen DynaMag-96 Side Skirted (Invitrogen, Cat. #12027) — for skirted
PCR plates
° Promega MagnaBot® II Magnetic Separation Device (Promega,
Cat. #V8351) — for PCR plates
- AlumaSeal II foil seals — for resealing the adaptor plate in the event that not
all adaptor wells are used at one time (Excel Scientific, Cat. #AFS-25)
- Disposable gloves
- Kimwipes
- Ice bucket
- Cleaning solutions such as DNA-OFF™ (MP Biomedicals, Cat. #QD0500)
To Order
• Affymetrix, Inc., www.affymetrix.com
• Beckman Coulter, www.beckmancoulter.com
• Covaris, www.covarisinc.com
• Excel Scientific, www.excelscientific.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
5 Ovation Rapid DR Multiplex System 1–96
III. Planning the Experiment
A. Input DNA Requirements
The Ovation Rapid DR Multiplex System 1–96 is designed to work with as little as
100–500 ng of fragmented genomic dsDNA or ds-cDNA. When using ds-cDNA gen-
erated by NuGEN’s Ovation RNA-Seq System V2 (Part No. 7102) or Ovation RNA-Seq
FFPE System (Part No. 7150), use at least 500 ng – 1 µg of of ds-cDNA. DNA samples
must be free of contaminating proteins, RNA, organic solvents (including phenol and
ethanol) and salts. Use of a commercially available system for DNA/cDNA isolation
is recommended. The A260:A280 ratio for DNA samples should be in excess of 1.8.
Use of DNA samples with lower ratios may result in low library yield.
B. Using the Ovation Rapid DR Multiplex System 1–96 on
Illumina NGS Platforms
The Ovation Rapid DR Multiplex System 1–96 uses a dedicated read (second sequenc-
ing primer) strategy, as shown in Figure 2.
Figure 2. Dedicated read (DR) barcoding strategy.
Dedicated Read
Barcode Design
Illumina
Standard
Seq Primer
Library Insert
Illumina
Index
Seq Primer
Barcode
Flow cell surface
The Ovation Rapid DR Multiplex System 1–96 uses 8-base barcodes and the same
approach to multiplexing found in the standard Illumina method. The resulting
libraries should be sequenced using the Illumina protocol for multiplex sequencing
for 8-base barcodes (total of 9 cycles). The DR barcode sequences can be found by
clicking the barcodes link on the Ovation Rapid DR Multiplex System 1–96 product
page at www.nugen.com and must be entered into the Illumina software prior to the
analysis.
6 Ovation Rapid DR Multiplex System 1–96
III. Planning the Experiment
C. Working with the NGS DR Adaptor Plate 1–96
The NGS DR Adaptor Plate 1–96 contains 96 ligation adaptor mixes, each with a
unique eight-base barcode. Each well of the 96-well plate contains sufficient volume
for preparation of a single library. The NGS DR Adaptor Plate 1–96 is sealed with a foil
seal designed to provide for airtight storage. Each well is designed for a single use.
Prior to thawing the adaptor plate, spin it for 5 to 10 minutes at room temperature at
~1000 X g in a centrifuge with a rotor appropriate for microwell plates. This will allow
the plate to thaw while spinning and help to minimize cross-contamination between
wells. To use only a portion of the plate, remove the seal covering only the portion of
the plate to be used. The remaining wells of the plate should remain sealed for use at a
later date. Used wells can be covered with a new foil seal (e.g., AlumaSeal II) to prevent
any remaining adaptor-containing liquid from contaminating future reactions.
D. Library Quantification
Libraries created using Ovation Rapid DR Multiplex System 1–96 must be quantified
using qPCR. These libraries cannot be accurately quantified using other means. We rec-
ommend using KAPA Library Quantification kits from KAPA Biosystems for quantifica-
tion. See Section V. Quantitative and Qualitative Assessment of the Library for details.
E. Amplified Library Storage
Amplified libraries may be stored at –20°C.
F. Data Analysis and Parsing Multiplex Libraries
Data analysis for next generation sequencing is an evolving field and the number of
available analysis strategies and software tools is growing rapidly. The specific analysis
workflow for a given experiment will depend on many variables, including the type of
sequencing application (DNA-Seq, RNA-Seq, etc.) and the goals of the particular study.
Follow the recommendations in the Illumina technical support documentation on
parsing barcodes. The sequences of the Ovation Rapid DR Multiplex System 1–96
barcodes must be entered prior to parsing. These sequences can be found by clicking
the barcodes link in the left hand menu of the Ovation Rapid DR Multiplex System
1–96 product page at www.nugen.com. Once the data have been parsed according to
sample, additional sample-specific data analysis may be conducted according to the
requirements of the experiment.
7 Ovation Rapid DR Multiplex System 1–96