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商品編號


13000-1



品牌


Lucigen



公司


Lucigen



公司分類(lèi)


NGS Products






商品信息

Close and finish your microbial genome with two libraries


Flex
IBL
e 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 imposs
IBL
e 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








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




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 par
ADI
gm 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


?

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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 poss
IBL
e.? 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



?



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


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
compat
IBL
e).? 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.