NxSeq® Long Mate Pair Library Kit and Index Kit

NxSeq® Long Mate Pair Library Kit and Index Kit


货号

13100-1


规格

10 libraries + 12 indices (5 libraries each)


存储温度

-20º C


产品详情


NxSeq® Long Mate Pair Library Kit

产品说明书

使用两个文库获得微生物基因组完成图

  • 灵活的插入片段大小:基于凝胶选择的方法20 kb,无需凝胶的方法< 8 kb
  • 通过现有的Illumina测序仪,利用20 kb构建流程可以得到微生物基因组完成图
  • 适用于二代测序的基因组重头组装、环化和完成、染色体重排检测、单体型分析,细菌人工染色体(BAC)测序
  • 增加二代测序组装片段的N50长度,获得更大的组装片段

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

更准确的组装以获得基因组完成图

由于重复序列的存在,使得contig的方向和顺序很难确认,因此片段化的文库无法组装成基因组。长的序列读长(通过mate pair文库、jumping文库或informative mate pairs获得)可以与片段化文库结合,正确组装二代测序数据成为更大的框架(scaffold),有利于基因组完成图的获得。加入mate pair文库可以更低成本的获得更好的基因组拼接;对于小的基因组有效减少额外的人工测序。

Figure 1: De novo assembly and closing Thermus aquaticus

  JGI Permanent
Draft Genome
Fragment library
NxSeq® Mate Pairs
+ fragment library
Manually finished
Thermus aquaticusgenome
# 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).


如何实现的?

通过高效的mate pair文库构建技术,Lucigen创造了新的长片段技术典范。基因组DNA被打断成期望的大小(2-8 kb基于磁珠分选,10-20 kb基于凝胶分选),末端补平,加A尾,片段分选前连接到barcode接头上。插入序列通过加密的Chimera Code™序列被连接到唯一的multiplex coupler上。然后,样品通过外切核酸酶处理,移除不需要的DNA;最后通过选择的内切酶消化产生正确大小的双标记片段。加入Junction Code接头并进行环化前,利用生物素捕获移除非目的DNA片段。然后,文库经PCR扩增并通过Illumina测序仪进行测序。

Figure 4.  NxSeq Long Mate Pair Library Workflow


Chimera Code™ 序列

Lucigen的Chimera Code序列(专利申请中)是获得高效真实mate paris的关键;最终使得准确的组装成为可能。软件分析最终序列并过滤掉文库制备过程中导致的假阳性pair。最终,大部分文库达到>90% mate pair有效率。


Figure 5. Chimeric Read Detection


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
 

插入片段大小灵活-自己选择文库片段大小

NxSeq Long Mate Pair Library Kit试剂盒可以适用于满足你需要的广泛插入片段大小。磁珠分选方案可使文库达到8 kb插入片段大小,而凝胶分选流程可以兼容10-20 kb插入片段大小。你的mate pairs片段大小结果是严格控制的,使得生物信息学可以准确的进行组装。

Figure 7. Long Mate Pair Libraries

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.


多文库制备的序列索引

想一次制备12个文库?Lucigen的NxSeq Long Mate Pair Library Index kit提供了12种不同带有不同序列索引的扩增引物对(与Illumina兼容)。请查询订购信息获取更多细节。

NxSeq分析脚本和数据集

为了对Illumina测序数据进行分析,需要首先运行脚本来确认Chimera Code 和Junction Code序列,并在组装前将这些序列过滤掉。这些脚本以及实例样本数据集可以从我们的网站找到。

NxSeq基因组环化服务

如果你要构建一个NxSeq Long Mate Pair文库,但不想亲自动手,你可以联系我们的基因组定制服务团队。我们可以Lucigen公司的很多服务对提供免费报价。

  • Ordering

  • Specifications

  • Manuals

  • Resources

Contact your local distributor for pricing
Product Description Size Cat. No.
NxSeq® Long Mate Pair Library Kit   10 libraries 1 13000-1
NxSeq® Long Mate Pair Library Kit and Index Kit   10 libraries1 + 
12 indices (5 libraries each)
13100-1
NxSeq® Long Mate Pair Index Kit   12 indices (5 libraries each) 13200-1
NxSeq® Long Mate Pair Library Kit, Box 1   10 libraries1 13300-1
NxSeq® Long Mate Pair Library Kit, Box 2   10 libraries1 13400-1
ORDER INFORMATION

本产品的全部试剂和组分可以参照我们的用户手册。The NxSeq Long Mate Pair Library kit试剂盒包含两个盒子,可以分开购买。Box 1含有末端修复、片段加尾、连接酶、内部接头序列的全部组分。Box 2含有连接到coupler的试剂及Junction Code™序列、核酸外切酶、生物素捕获及扩增所需的试剂。

对于12个分开的index引物组中的每个引物组,NxSeq Long Mate Pair Library Index kit均含有5个反应所需试剂。该试剂盒可以与文库构建试剂盒同时购买,或单独购买。

仅供研究使用。不用于人体实验或诊断。

试剂盒可供构建10个8kb或小于8kb片段的文库。更大片段的文库,10-20kb,会使用更多的试剂,每个试剂盒可构建的文库个数将少于10个。构建10-20kb插入片段的mate pair文库可参考SP001: NxSeq 20 kb Mate Pair Protocol.

* Discount valid thru September 30, 2015.