Lucigen NxSeq AmpFREE DNA Lib Kit Dec2015

Other MiSeq Genome Sequencing Examples Highly Mappable Reads
Staphylococcus
Genome size, GC Raw reads Mapped reads Read length Total bases Genome fraction Avg. coverage Max coverage 2,821,361 33%GC 1,260,836 1,174,111 (93.12%) 148.8 bp 174,694,261 0.97 62X 172X
Optimized reagents and protocol
• Highest efficiency end-repair, A-tailing & ligation = more complex libraries and better sequencing coverage
Low DNA input with no amplification
Minimal handson time too!
Only 4 Key Components Simplify Usage and Affordable Pricing Reduces Costs
Product NxSeq® AmpFREE Low DNA Library Kit
Cat. No. 14000-1 14000-2
75ng
75ng
75ng
0
0
0
Swift
5
5
5
NxSeq
MiSeq Human Genome Sequencing Example >95% of Reads Map to the Genome
75 ng Human DNA Input Library Genome size, GC ~3 Gbp 45% GC Raw reads 3,131,114 Mapped reads 2,979,237 (95.15%) Read length 148.9 bp Total bases 443,767,447 Genome fraction 0.11 Avg. coverage 0.15X
75 ng input DNA More than enough library
Sufficient Library DNA Generated from Only 75 ng Input DNA
* MiSeq requires input of library DNA at 2 nM in 5 µL volume
Results: • On average, generated >1.7X more concentrated library than needed to add to the MiSeq • Sufficient yield from each library at a high enough concentration for multiple sequencing runs
25 20 15 20 15
E. coli K12 (~50% GC)
20
15 10
20 15 10
10
Illumina Illumina NxSeq NxSeq Kapa Kapa Swift Swift
5 0
10
Illumina
Illumina
NxSeq
Kapa
Kapa
250ng
Swift
1µg
1µg
1µg
250ng
100ng
250ng
100ng
250ng
100ng
1µg
• •
Vendor suggested fragmented DNA input Ran 5 µL of 2 nM samples normalized using Bioanalyzer and Qubit
100ng
75ng
Lucigen NxSeq 75 ng input
Kapa Hyper Prep Kit 250 ng input Swift Biosciences Accel-NGS 2S DNA Kit 100 ng input
• •
45.5 39 29.5 28
13
40
26.5 33
18
25.5
49
-
45
45
Highly Efficient Library Construction Greater Percentage of Identifiable Clusters
Human (45% GC)
% Clusters Identified
Rhodobacter (68% GC)
25
Staphylococcus (24% GC)
$150
$150
$3
$3
Library Kit Components: Enzyme Mix, 2X Buffer, Ligase, Elution Buffer
Summary
Highest efficiency PCR-free kit enables • Use of very small amounts of input DNA (75 ng) without the need for amplification • Better sequencing coverage and flow cell utilization Rapid protocol saves valuable time Affordable cost/adaptor costs are significantly lower than Illumina kits thus extending your budget
Total Library Yield in 20 µL Library -Input Human - 75 ng Staph – 75 ng Rhodo – 75 ng ng 28.6 29.8 23.2 Library Concentration ng/µL 1.43 1.49 1.16 nM 3.61 4.33 2.83 Volume of Library required to create 5 µL of a 2nM stock 2.77 µL 2.31 µL 3.53 µL
Size (rxn) 12 48 12 x 4 (12 adaptors, 4 rxn ea.)
US 2016 List Price
$240 $921
Price Per Rxn
$20 $19
NxSeq® Adaptors, Box 1
NxSeq® Adaptors, Box 2
14300-1
14400-1
NxSeq® AmpFREE™ Low DNA Library Kit
High Efficiency DNA Fragment Library Prep for Illumina NGS Instruments
November, 2015

NxSeq® AmpFREE™ Low DNA Library Kit Minimal DNA Input, No Amplification Bias
• 75 ng sheared DNA input = usable with limiting samples • No PCR = no amplification bias introduced
Fast and efficient
• DNA libraries in 2 hours = saves time and gets on the sequencer faster
Rhodobacter
4,602,977 69%GC 3,900,174 3,613,165 (92.64%) 149.6 bp 540,403,552 1.00 117X 364X
Used 75 ng sheared input DNA for fragment library prep
Fastest Protocol – Only ~2 Hours Total
Highly Efficient Library Construction PrLibrary ID Human Ave Illumina TruSeq 1 µg input 7 49 35 13 42 24 10 1 1 35 17 Staphylococcus Ave 1 37 22.5 4 60 31 12 59 41 8 43 39 Rhodobacter E. coli K12 Ave 10 51 40
Two libraries prepped per kit/organism using the manufacturer’s recommended input amounts. Adaptor ligation efficiency measured by qPCR using an amplified library standard.
Automation friendly
• Easily automate library prep with single tube reaction format
Minimal DNA Input Produces Plenty of Library Ready to Load on the Sequencer