Nextera Mate Pair Sample Preparation Kit de novo

Data Sheet: Sequencing
Nextera® Mate Pair Sample Preparation Kit
An optimized sample preparation method for long-insert libraries that empower de novo
sequencing and detection of structural variants.
Highlights
Figure 1: Nextera Mate Pair Workflow
• Fast and Simple Mate Pair Preparation
Go from DNA to libraries in less than two days with a simple
tagmentation reaction and low DNA input
• Dual Protocol Flexibility
Gel-free and gel-plus protocols enable a range
of applications, including de novo assembly and structural
variation detection
• Premier Data Quality
Highly diverse libraries maximize data yield
• End-to-End Mate Pair Solution
Conveniently bundled kit includes reagents and indices
for efficient mate pair preparation
Introduction
Mate pair library sequencing enables the generation of long-insert
paired-end libraries. The Nextera Mate Pair Sample Preparation kit
offers two methods, gel-free and gel-plus, to support a variety of
applications and input requirements. The robust, low-input,
gel-free protocol yields high-diversity libraries that enable deeper
library sequencing. The size-selection process of the gel-plus protocol
generates fragments with a tight size distribution for structural variation
detection. Libraries prepared with the gel-plus protocol also provide
sequence information for larger repeat regions, empowering de novo
genome assembly.
B
Dual Protocol Flexibility
The flexibility of Nextera Mate Pair sample preparation stems from the
availability of two different size-selection options (Table 1). The gelfree protocol, which requires only 1 μg DNA, provides highly diverse
mate pair libraries with a broad range of fragment sizes (Figure 2A).
This protocol is ideal for routine de novo assembly of simple bacterial
genomes, or for the robust generation of mate pair data on samples
with limited DNA. The gel-free protocol offers a faster, simplified option
with a lower DNA input requirement to streamline mate pair studies.
B
B
B
Genomic DNA (blue) is tagmented with a Mate Pair Tagment Enzyme, which attaches
a biotinylated junction adapter (green) to both ends of the tagmented molecule.
B
B
B
B
The tagmented DNA molecules are then circularized and the ends of the genomic
fragment are linked by two copies of the biotin junction adapter.
B
B
B
B
B
B
Circularized molecules are then re-fragmented yielding smaller fragments.
Sub-fragments containing the original junction are enriched via the biotin tag (B)
in the junction adapter.
Simplified Mate Pair Workflow
The Nextera Mate Pair protocol provides a simple mate pair workflow
(Figure 1) for the preparation of sequencing-ready libraries in less than
two days. Master-mixed TruSeq DNA Sample Preparation reagents
reduce the number of assay steps, thereby reducing hands-on time
to as little as three hours. The Nextera “tagmentation” reaction utilizes
a specially engineered transposome, the Mate Pair Tagment Enzyme,
to simultaneously fragment and tag the DNA sample. This simplified
method only biotinylates DNA molecules at the sites of fragmentation,
avoiding troublesome internal biotinylation.
B B
B B
B
B
+
B
B
After End Repair and A-Tailing, TruSeq DNA adapters (grey and purple) are
then added, enabling amplification and sequencing.
The Nextera Mate Pair Sample Preparation kit has a simple workflow
that enables library preparation in less than two days and supports a
range of fragment sizes from 2–15 Kb.
The gel-plus protocol, which requires 4 μg DNA and standard agarose
gels or SAGE Pippin Prep gels1, offers a more stringent size selection
process. The gel-plus protocol produces libraries with tighter size
distributions to facilitate structural variation detection (Figures 2B and
3). Greater control over fragment sizes is ideal for more challenging
mate pair applications, such as de novo assembly of complex
genomes and structural variation detection.
Data Sheet: Sequencing
Figure 2: Fragment Size Distribution with Dual Protocols
A
B
1.2
0.4
0.35
1
Frequency
0.3
0.8
0.25
0.6
0.2
0.15
A
0.4
0.1
0.2
0.05
B
1.2
0.4
0.35
0
0
0
1000
3000
5000
7000
9000
11000
13000
15000
0
Frequency
Fragment Size (bp)
1
1
0.3
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
0.25
0.8
Fragment Size (bp)
0.6
0.2
Panel A shows the fragment size distribution of an E. coli mate pair library prepared using the 0.15
Nextera Mate Pair gel-free protocol, resulting in a broad fragment size
0.9
distribution.
Panel B shows the fragment size distribution of an E. coli mate pair library generated with the Nextera Mate Pair gel-plus protocol, using automated size
0.1
selection with the Pippin Prep platform.
0.8
0.05
Frequency
0.7
0
Highly0.5Diverse Libraries
2000
4000
3000
5000
7000
9000
11000
6000
8000
10000
12000
The Nextera Mate Pair Sample Preparation
Fragment SizeKit
(bp)also provides
identifiable junction sequences
that
mark
fragment
ends, greatly
3 Kb
5 Kb
8 Kb
simplifying informatics analysis. The presence of searchable junction
sequences allows for accurate fragment identification and enables
sequencing of longer read lengths, as mate pair junctions can be
precisely identified and trimmed accordingly.
Mate Pair Preparation Solution
In addition to Nextera Mate Pair reagents, the comprehensive Nextera
kit contains TruSeq DNA sample preparation reagents and indices.
Nextera Mate Pair tagmentation and circularization are followed
by TruSeq on-bead reactions (Figure 1), simplifying the purification
workflow and reducing sample loss. This integrated solution
streamlines the sample preparation workflow, maximizing sequencing
efficiency with more samples per lane and enabling rapid multiplexed
sequencing of small genomes. The Nextera Mate Pair kit is compatible
with TruSeq DNA Sample Preparation adapter indexing, supporting 12
indices per kit for a scalable experimental approach. With all necessary
reagents included in one convenient, cost-effective bundle, the
Nextera Mate Pair Sample Preparation kit is the all-in-one solution for
faster and simpler mate pair preparation.
13000
15000
1
0.9
0.8
0.7
Frequency
0
1000
Figure 3: Fragment Size Distribution
Fragment Size (bp)
0.4
The Nextera
tagmentation reaction drives the creation of highly diverse
libraries0.3
(Table 2) that are compatible with all Illumina sequencing
systems.
Library diversity is defined as the number of unique
0.2
fragments in a given library. The Nextera Mate Pair protocol allows for
0.1
the creation of millions of unique fragments, increasing library diversity
0 fewer duplicate reads and yield greater amounts of data.
to generate
0.2
0
0
0.6
0.4
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2000
4000
6000
8000
10000
12000
Fragment Size (bp)
3 Kb
5 Kb
8 Kb
This plot shows fragment size distributions of three E. coli mate pair libraries
(3 Kb, 5 Kb, and 8 Kb) created from the same tagmentation reaction. These
distributions were generated following the Nextera Mate Pair gel-plus
protocol and agarose gel size-selection procedure.
0
1000 2
Data Sheet: Sequencing
Ordering Information
Table 1: Nextera Mate Pair Protocols
Protocol
DNA
Input
Number
of
Samples
Size
Selections
Per Sample
Number of
Libraries
Gel-Free
1 μg
48
N/A
48
Gel-Plus with
Pippin Prep
size selection
4 μg
12
1
12
Gel-Plus
with agarose
size selection
4 μg
12
Up to 4
Up to 48
Catalog No.
FC-132-1001
This kit contains Nextera Mate Pair reagents and TruSeq reagents and indices.
Summary
Table 2: Nextera Mate Pair Library Diversity*
Preparation
Product
Nextera Mate Pair Sample Preparation Kit
With a fast and easy workflow, the Nextera Mate Pair Sample
Preparation kit allows the construction of high-quality sequencing
libraries in less than two days. The gel-free and gel-plus options
provide flexibility for a variety of specific applications. Transposomemediated tagmentation, identifiable junction sequences, and indexing
capability make Nextera Mate Pair Sample Preparation the simplest,
easiest solution for mate pair applications.
Input DNA
Fragment Size
Diversity**
Nextera
Mate Pair
Gel-Free
References
1 μg
~2–15 Kb
860 million
1. www.sagescience.com/products/pippin-prep
Nextera
Mate Pair
Gel-Plus
4 μg
~2–4 Kb
568 million
Nextera
Mate Pair
Gel-Plus
4 μg
~5–7 Kb
396 million
Nextera
Mate Pair
Gel-Plus
4 μg
~6–10 Kb
102 million
2. Lander ES and MS Waterman. (1988) Genomic mapping by fingerprinting
random clones: a mathematical analysis. Genomics 2(3): 231–9.
www.ncbi.nlm.nih.gov/pubmed/3294162
*This table demonstrates example diversity values, with diversity reported
in number of unique fragments. Actual diversities achieved with the
Nextera Mate Pair kit may vary and are dependent on several factors,
including DNA input quantity, DNA quality, and precise execution of
the protocol.
**Library diversity was calculated from the number of unique read
pairs observed in a data set, using a method based on the
Lander-Waterman equation.2
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Pub. No. 770-2012-052 Current as of 20 December 2012