您做新一代测序的原因是您需要大数据。为此，你需要把每一个样本都建成好的文库来获得多的信息量。那么为什么不选择一款好的DNA文库构建盒子来取代您所花费的时间，资源和昂贵的测序试剂。
The NxSeq® AmpFREE Low DNA Library Kit 可以让您建立好的文库。我们优化了操作步骤的每一步确保在illumina测序仪上发挥优的表现。此外盒子仅需要输入剪切DNA的量为75 ng。使用简化，容易的操作步骤在2小时的内构建出文库。
More Sequenceable DNA Fragments per Library = More Data
Figure 1. Percentage of library DNA with correctly ligated adaptors measured by qPCR. Duplicate libraries were prepped per kit/organism (Human, Staphylococcus aureus, Rhodobacter sphaeroides (1 library only), and E. coli) according to the manufacturer’s recommended input amounts and protocols. Adaptor ligation efficiency was measured by qPCR using the KAPA Library Quantification Kit (Complete ROX Low, cat #KK4873) and matching amplified library as an internal standard.
Figure 2a. Number of sequencing reads generated per library after multiplexing and running on a MiSeq Instrument. DNA fragment libraries were prepped in parallel for each kit/organism according to the manufacturer’s recommended input amounts and protocols. Libraries were quantitated and normalized to 2 nM using the Bioanalyzer (size) and Qubit Fluorometer (amount). Equimolar amounts of each library were multiplexed and sequenced with a single MiSeq run using 2 ×150 bp chemistry. The number of sequencing reads obtained are shown as well as the percent reduction (%) in total reads compared to the appropriate NxSeq AmpFREE Kit results.
More Proof with Challenging FFPE Samples
Figure 2b. Number of sequencing reads generated from matching normal and FFPE gDNA sample libraries. DNA fragment libraries were prepped using the two indicated kits according to the manufacturer’s recommended input amounts and protocols. Libraries were constructed from normal gDNA (Biochain, Cat. No. D1234142-S02) and DNA extracted from a matching FFPE human kidney tissue (Biochain Cat. No. T2234142-S02) using the Qiagen AllPrep DNA/RNA FFPE Kit. The gDNA samples were sheared to ~250 bp before starting library construction. Final libraries were quantitated and normalized to 2 nM using the Bioanalyzer (size) and Qubit Fluorometer (amount). Equimolar amounts of each library were multiplexed and sequenced with a single MiSeq run using 2 × 150 bp chemistry. The number of sequencing reads obtained are shown as well as the percent reduction (%) in total and mapped reads compared to the corresponding NxSeq AmpFREE Kit results using 75 ng of input DNA.
Sequencing Stat
Rhodobacter
Genome size, GC percentage
~3 Gbp 45% GC
2,821,361 33% GC
4,602,977 69% GC
Raw reads
3,131,114
1,260,836
3,900,174
Mapped reads
2,979,237 (95.15%)
1,174,111 (93.12%)
3,613,165 (92.64%)
Read length
148.9 bp
148.8 bp
149.6 bp
Total bases
443,767,447
174,694,261
540,403,552
Genome fraction
0.11
0.97
1.00
Avg. coverage
0.15X
62X
117X
Figure 3. Representative gDNA sequencing stats from three different organisms. Genomic DNA fragment libraries were generated using the NxSeq AmpFREE Low DNA Library Kit using 75 ng of sheared gDNA input from three organisms (human, Staphylococcus aureus, and Rhodobacter sphaeroides). The final libraries were quantitated and normalized to 2 nM final concentrations using the Bioanalyzer and Qubit fluorometer, and 5 µL of each library was run on a MiSeq using 2 x 150 bp chemistry and analyzed.
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