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公司介绍
美国Lucigen公司,自1998年成立至今,一直致力于生命科学领域相关科研产品的研究与开发,在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品,包括:CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等。Lucigen公司凭借其独到的产品技术,过硬的产品质量,...
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Lucigen/NxSeq® Long Mate Pair Library Kit, Box 2/13400-1/10 libraries1

  • 产品编号: 13400-1
  • 美  元  价: $2281.00
  • 会  员  价: 待定
  • 品       牌: Lucigen
  • 产       地: 美国
  • 公       司: Lucigen
  • 产品分类: ref="." class="index">首页 > Lucigen/NxSeq® Long Mate Pair Library Kit, Box 2/13400-1/10 libraries1
  • 公司分类: NGS Products
Lucigen/NxSeq® Long Mate Pair Library Kit, Box 2/13400-1/10 libraries1
  • Lucigen/NxSeq® Long Mate Pair Library Kit, Box 2/13400-1/10 libraries1
商品介绍

Close and finish your microbial genome with two libraries

  • FlexIBLe insert sizes: gel-based 20 kb and gel-free < 8 kb
  • Enable microbial genome closure using 20 kb protocol with your existing Illumina sequencer
  • Ideal for NGS de novo genome assembly, closure and finishing, chromosomal rearrangement detection, haplotyping, and BAC sequencing
  • Increased N50's and larger scaffolds from your assembly in Next Gen Sequencing

Table of Contents

  • More accurate assemblies to close and finish your genome
  • How does it work?
  • Chimera Code™ sequences
  • Insert size flexibility
  • Indices for multiplexing libraries
  • NxSeq Scripts and Sample Data Set
  • NxSeq Genome Closure Services

More accurate assemblies to close and finish your genome.

Fragment libraries are not sufficient to fully assemble genomes due to repetitive elements that make the correct order and orientation of contigs impossIBLe to determine.  Long span read data (obtained through mate pair libraries, jumping libraries, or informative mate pairs) can be combined with fragment libraries to properly assemble next generation sequencing data into large scaffolds, enabling easier genome closure and finishing.  The addition of mate pair libraries can make cost-effective genome closure a reality, with limited manual sequencing required for small genomes.


Figure 1: De novo assembly and closing Thermus aquaticus

  JGI Permanent
Draft Genome
Fragment library
NxSeq® Mate Pairs
+ fragment library
Manually finished
Thermus aquaticus genome
# Contigs > 500 bp 22 54 22 NA
Contig N50 106 kb 79 kb 144 kb NA
Max contig achieved 343,213 167,687 260,181 NA
Genome scaffolds > 5kb 0 0 1 1

Max scaffold achieved

343,213 0 2,161,678 2,158,963
Genome size 2,338,193 2,256,923 2,161,678 2,338,240
Plasmid scaffolds ? 0 2 4
Plasmid sizes ? NA 14.5 kb, 70.3 kb 14,047 bp, 16,597 bp
78,727 bp, and 69,906 bp
 

Figure 2: Single Scaffold Assembly of E. coli K12
Fragment + 8kb Mate Pair Library

Fragment library

5,001,861 reads – 4,656,638 mappable
Assembled 2.5 M reads in SPAdes with K45

  • Contigs > 1kb:  163
  • Contigs > 500 bp:  184
  • N50:  56,903
  • Max contig: 179,213 bp

8 kb NxSeq library

Megaruptor sheared

  • Raw reads:  6,909,356 
  • True mate pairs:  3,288,275 (48% of raw)
  • Mate pair distance:  8,310 bp

K12 Scaffold

De novo assembly of E. coli K12 genome. 2.5M fragment reads were assembled de novo into 163 contigs over 1 kb by SPAdes 3.1. Scaffolding was performed with commercial software using 3.2M 8 kb mate pairs. The single scaffold was compared to a reference  genome with Mauve 2.3.1.

 

Figure 3: Assembly of Repeat-Rich Mouse BACs
Assembly Forms One 171kb Scaffold

Assembly Repeat-Rich Mouse BACs

Sequence assembly for two repeat-rich mouse BACs. The sequences were assembled with DNAStar software using Ion Torrent 400 bp fragments and 5 kb NxSeq sequence data. Despite having over 50% repeat sequence, two BACs were each assembled into single scaffolds of 171 kb (shown) and 143 kb (not shown).

 

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How does it work?

Lucigen has created a new parADIgm in long span read technology via highly efficient mate pair library prep technology.  Genomic DNA is sheared to the desired size (2-8 kb for bead-based methods and 10-20 kb for gel-based sizing methods), end repaired, A-tailed and ligated to barcode adaptors prior to size selection. The insert is ligated to a unique multiplex coupler with encrypted Chimera Code™ sequences. Samples are then treated with exonuclease to remove unwanted DNA, and finally digested with a selection of endonucleases to produce the correct sized di-tags. Biotin capture allows for the removal of unwanted DNA fragments prior to the addition of a Junction Code adaptor and re-circularization.  Libraries are then PCR amplified and sequenced on an Illumina sequencer.

Figure 4.  NxSeq Long Mate Pair Library Workflow

NxSeq Long Mate Pair Workflow

 

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Chimera Code™ Sequences

Lucigen's patent-pending Chimera Code sequences are the key to achieving ultra-high frequencies of true mate pairs, ensuring the most accurate assembly possIBLe.  Software analysis of final sequences filters out false mate pairs formed by chimeras during the library prep process.  As a result, most libraries achieve >90% true mate pair efficiency.


Figure 5. Chimeric Read Detection
Chimera Code Sequence Prevents False Mates

 

Figure 6.

  E. coli DH10B
2kb
E. coli DH10B
5kb
E. coli DH10B
8kb
Raw Reads 6,377,792 5,995,974 6,851,682
Total Mates 2,167,286 2,242,930 3,091,359
True Mate Pairs 2,071,267
(96%)
2,094,413
(93%)
2,938,426
(95%)
Chimeric Reads 96,019
(4%)
148,517
(7%)
152,933
(5%)

Avg. Read Length (after split)

170 b 161 b 159 b
Total Mate Pair Bases 352,115,390 337,200,493 467,209,734
Mapped Mate Pair Distance 2,543 5,145 6,191
 

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Insert Size Flexibility – You Choose Your Library Size

The NxSeq Long Mate Pair Library Kit can accommodate a wide range of insert sizes to fit your needs.  Bead-based, gel-free fragment sizing protocols enable libraries up to 8 kb insert size, while gel-based sizing protocols will accommodate 10-20 kb insert size.
The result is tight sizing of your mate pairs, enabling accurate and complete bioinformatic assembly.

Figure 7. Long Mate Pair Libraries

Long Mate Pair Libiraries

An 8 kb NxSeq Long Mate Pair library was constructed using bead-based, gel-free methods, and a 10-20 kb mate pair library was constructed using gel isolation.  Resulting true mate pairs were mapped against the respective reference genome to determine the resulting mate pair distances.

 

 

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Indices for
Multiplexing Libraries

Want to multiplex up to 12 libraries at one time?  Lucigen offers the NxSeq Long Mate Pair Library Index kit with 12 different indexed amplification primer sets (Illumina compatIBLe).  See the ordering information tab for more details.

NxSeq Scripts &
Sample Data Set

To perform bioinformatic analysis of your Illumina runs, scripts must be run to confirm Chimera Code and Junction Code sequences as well as filter out these sequences prior to final assembly.  These scripts, along with a sample data set for trial analysis can be found here. 

NxSeq Genome
Closure Services

Would you like to have a NxSeq Long Mate Pair library, but don't want to do it yourself? Contact our Custom Genomic Services group and we'll provide a no-obligation quote for a range of services offered by Lucigen.

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ORDER INFORMATION

For a full list of reagents and components included in this product, refer to the user manual.  The NxSeq Long Mate Pair Library kit includes two boxes, each of which can be ordered separately.  Box 1 contains all reagents necessary for end repair and tailing of fragmented DNA, ligase, and an internal adapter sequence.  Box 2 contains reagents for ligation to the coupler and Junction Code™ sequence, exonuclease digestion, biotin capture, and amplification. 

The NxSeq Long Mate Pair Library Index kit contains 5 reactions each of 12 separate index primer sets, for a total of 60 index reactions.  The kit may be ordered in combination with the library kit or as a separate item.

For research use only.  Not for human or diagnostic use.

1 This kit contains the reagents necessary to generate 10 libraries of 8kb or less. Larger libraries, 10-20kb, will use more reagents and generate fewer libraries per kit.  Instructions to generate mate pair libraries using 10-20kb inserts are described in SP001: NxSeq 20 kb Mate Pair Protocol.

品牌介绍

美国Lucigen公司,自1998年成立至今,一直致力于生命科学领域相关科研产品的研究与开发,在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品,包括:CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等。Lucigen公司凭借其独到的产品技术,过硬的产品质量,良好的产品服务赢得了全球广大用户的信赖。

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