Accel-NGS™ 2S DNA Library Kit for Illumina

DNA Library Preparation for Next Generation Sequencing (NGS) on Illumina Platforms

Catalog No   Library Kit Reactions 
 DL-ILM2S-12 Accel-NGS 2S DNA Library Kit for Illumina 12
 DL-ILM2S-48 Accel-NGS 2S DNA Library Kit for Illumina 48


An Indexing Kit is required for complete functionality. Please select one of the following:

Catalog No Indexing Adapter Kit  Reactions 
SI-ILM2S-48A  2S Set A Indexing Kit (12 indices x 4 rxns each)  48
 SI-ILM2S-48B  2S Set B Indexing Kit (12 indices x 4 rxns each) 48
SI-ILM2S-96 2S Set A+B Indexing Kit (24 indices x 4 rxns each) 96
 DI-ILM2S-96  Dual Indexing Kit (20 indices x 96 combinations) 96
SP-ILM2S-12 2S Single Index Kit (a single Index, 12 rxns) 12
 XT-ILM2S-12 XT Compatibility Module (adapters and pre-hyb PCR primers) 12
 XT-ILM2S-48 XT Compatibility Module (adapters and pre-hyb PCR primers) 48

 

Improve DNA Sequencing Results by Covering More of the Genome

The Accel-NGS 2S DNA Library Kit for Illumina utilizes a proprietary adapter attachment chemistry which minimizes bias and supports inputs as low as 10 pg. This technology delivers a host of benefits, including:

  • PCR-free libraries from inputs as low as 10 ng
  • Linear yield performance for DNA inputs from 10 pg to 1 µg
  • Excellent efficiency, data quality and evenness of coverage
  • No base composition bias from AT-rich or GC-rich genomes
  • Compatible with the leading hybridization capture products

Accel-NGS 2S is the best choice for users seeking the most versatile, highest performing library prep kit. Expand your capabilities, run those difficult samples or add new services to a core lab.

Product Details

Accel-NGS 2S technology is based on proprietary molecular biology techniques and reagents from Swift Biosciences. These combine to produce a protocol for generating libraries that exhibit exceptional evenness of coverage from a wide dynamic range of input levels. The kit’s broad dynamic input range, more than five orders of magnitude, means that pre-library quantification is optional, further streamlining the workflow. Finally, the Accel-NGS 2S DNA Library Kit protocol is readily automatable for those with high throughput applications.

Supported DNA Sequencing Applications and Sample Types

  • Whole Genome Sequencing (WGS)
  • Exome / Hyb Capture
  • Circulating Cell-free DNA (cfDNA)
  • Targeted / Amplicon Sequencing
  • Metagenomics
  • ChIP-Seq
  • FFPE
Features Benefits
Broad input range from 10 pg to 1 µg Reduce concerns of proper quantification
PCR-free libraries from ≥ 10 ng inputs Minimizes sequence bias from PCR
Superior library complexity Generate more unique library molecules
Exceptional coverage of AT/GC-rich sequences More complete genome coverage
Readily automatable Process more samples per day

 

Accel-NGS 2S DNA Library Kit for Illumina Workflow

Each step is color-coded to make the protocol easy to follow.

A simple single tube “with bead” protocol is provided with the kit in order to streamline processing between reactions. As the clean-up beads are not provided with the kit, we recommend the SPRIselect Reagent Kit from Beckman Coulter.

Video: Using the Accel-NGS 2S DNA Library Kit

Click here to view the video

Performance with Microbial and Human gDNA on the Illumina Platform

The data below demonstrate the performance of the Accel-NGS 2S DNA Library Kit with microbial and human genomic DNA.

Accel-NGS 2S Produces Linear Yields from 10 pg to 1 µg

 Accel-NGS 2S chemistry is capable of processing a broad range of input quantities, including ultra-low input. Library yields across input quantities from 10 pg to 1 µg are linear. Unlike some competitors, Accel-NGS 2S does not require precise titration of input amounts.

Figure 1. Accel-NGS 2S library yields from different sources and quantities. Prepared library yields were determined by qPCR quantification for genomic DNA from human, E. coli, and S. aureus. Library yields were linear over a range of 5 orders of magnitude, from 10 pg to 1 µg.

 

Quality Sequence from FFPE with Accel-NGS 2S

DNA from FFPE samples is susceptible to substantial damage resulting from the fixation process that introduces cross-links and fragmentation; oxidation damage also accumulates during prolonged storage. This damage can greatly reduce the amount of usable DNA molecules for NGS library preparation, becoming a barrier to analysis. Accel-NGS 2S addresses the growing demand for a method that can convert a small amount of damaged DNA into a high quality NGS library.

Unlike many competitors’ products, which are not recommended or promoted for use with FFPE, Accel-NGS 2S chemistry can handle both damaged and short DNA fragments. This makes Accel-NGS 2S ideally suited for clinical samples such as FFPE or plasma**, and it produces exceptional results even from low input quantities.

Figure 2. Accel-NGS 2S libraries were constructed from 10 ng (left) or 1 ng (right) of DNA from a clinical FFPE sample followed by 9 and 12 cycles of amplification, respectively. Sequencing was performed on an Illumina HiSeq 2500 in rapid run mode. Picard CollectGcBiasMetrics (picard.sourceforge.net) demonstrated evenness of coverage regardless of input amount and number of PCR cycles.

 

Get Higher Library Yields – Even with FFPE DNA

Head-to-head evaluations of library yields show that Accel-NGS 2S produces consistently higher yields than a competitor’s kit. This performance advantage was not diminished by the quality of the input DNA; Accel-NGS 2S outperformed on both high quality DNA as well as lower quality DNA extracted from FFPE. These results demonstrate that Accel-NGS 2S is more robust and versatile for a variety of applications.

Figure 3. Fold increase in library yield for Accel-NGS 2S versus kit U from company N was determined using the same DNA samples (high quality gDNA and low quality gDNA extracted from FFPE). Prepared libraries were quantified by qPCR, where yields were determined prior to performing a library amplification step, and compared by paired Student’s t-test. Accel-NGS 2S kit produced an average 2.9 ± 0.5 fold increase in yield as compared to kit U. Accel-NGS 2S libraries constructed using low quality DNA extracted from FFPE samples showed an average 3.5 ± 1.5 fold increase in yield compared to kit U. *Indicates statistically significant P-values = 0.024 and P <0.0001, respectively. HapMap DNA NA12878 was obtained from Coriell and E. coli DNA was obtained from Affymetrix (N = 8). 22 patient FFPE samples were obtained from Spectrum Health Biorepository and Baylor College of Medicine.

 

Boost Data Output and Quality at Ultra-Low Input

Obtaining high quality sequence information from ultra-low starting material has been a challenge. Two difficulties associated with ultra-low input library generation are PCR amplification bias and generation of adapter dimers. PCR amplification bias reduces the complexity of the library while the presence of adapter dimers takes up valuable space on the sequencing flow cell, and both result in data loss.The unique chemistry of Accel-NGS 2S library preparation allows high quality sequencing of ultra-low input DNA samples by virtually eliminating the formation of adapter dimers. This technology enables researchers to obtain more usable sequence from less sample. This has the secondary consequence of reducing the amount of PCR amplification required to minimize bias and produce a more even sequence coverage.Side-by-side comparison of Accel-NGS 2S versus libraries produced by either kit Na or U of competitors showed superior performance at all inputs (1 ng, 0.1 ng and 0.01 ng DNA). Strikingly, at 0.01 ng input, adapter dimers made up 36% and 61% of the libraries constructed with kits Na and U, respectively, while adapter dimers were a mere 0.3% of the library constructed with Accel-NGS 2S.

Figure 4. Accel-NGS 2S was compared to commercially available kit Na from company I and kit U from company N, which were tested “off label” for ultra-low input performance, using the minimum amount of amplification necessary for sequencing. To quantify data loss, the percentage of adapter dimers was determined for each sample. Sequencing was performed with Illumina MiSeq V2 reagents. Data was analyzed using Picard (picard.sourceforge.net) and a custom script. Accel-NGS 2S library adapter dimers were negligible and significantly below that seen in the competitor libraries.

 

Evenness of Coverage Over a Wide Input Range: E. coli

Multiple features of Accel-NGS 2S combine to produce even, unbiased coverage. This can be readily demonstrated by analyzing the WGS coverage vs. GC content and theoretical distribution of libraries made from E. coli. Accel-NGS 2S produces excellent data versus the competition, and equally important, there is no compromise in the data quality, irrespective of input ranging from 10 pg to 1 μg.

Figure 5. E. coli WGS libraries were constructed using Accel-NGS 2S, at 1 μg PCR-free or 10 pg with 15 cycles of PCR. Coverage was even across GC content and comparable to theoretical distribution, regardless of input. Similar results were observed for 100 pg with 12 cycles of PCR, 1 ng with 9 cycles of PCR, 10 ng with 6 cycles of PCR, and 100 ng PCR-free; data not shown. Libraries were also constructed using company I’s kit Na at its recommended 100 ng input with PCR, company N’s kit U at its recommended 10 ng input with PCR and company I’s Ne-XT kit used at its recommended 1 ng input with PCR. Sequencing was performed with Illumina MiSeq V2 reagents. Data was analyzed using BWA (Li and Durbin, 2010) and Picard (picard.sourceforge.net). In particular, note the unevenness of coverage for the Ne-XT kit versus Accel-NGS 2S.

 

Expect Even Coverage with Human DNA Sequencing

Accel-NGS 2S also offers outstanding evenness of coverage for larger, complex genomes such as human. This holds true for both PCR-free and libraries requring PCR amplification. This enables high quality libraries to be constructed from various inputs of human DNA without compromising the quality of the data.

Figure 6. Accel-NGS 2S libraries were constructed from Coriell HapMap NA12878 DNA, 100 ng PCR-free or 10 ng with 6 cycles of PCR. Sequencing was performed on an Illumina HiSeq 2500 in rapid run mode. Data was analyzed using BWA (Li and Durbin, 2010) and Picard CollectGcBiasMetrics (picard.sourceforge.net). The coverage vs. theoretical plot on the left panel demonstrates that libraries constructed from both 10 ng input with 6 cycles of PCR and 100 ng PCR-free show a comparable distribution to the theoretical. The right panel demonstrates that a library constructed from 100 ng PCR-free with Accel-NGS 2S provides even coverage across a range of GC content.

Validation on the Illumina MiSeq

The data below demonstrate the performance of the Accel-NGS 2S DNA Library Kit with microbial genomes of varying base composition that were prepared and sequenced on the Illumina MiSeq by a third party.

Comparable Coverage from 10 pg to 500 ng Input for AT-rich Genome

  • 10 pg to 500 ng of S. aureus genomic DNA (33% GC) was prepared using the Accel-NGS 2S DNA Library Kit. PCR was only used for inputs below 100 ng.
  • Left: Cumulative fraction of genome covered vs. depth of coverage, with theoretical perfect result (red) when entire genome is at normalized depth
  • Right: GC content of each read vs. theoretical in silico distribution and coverage

Conclusion. The Accel-NGS 2S DNA Library Kit produces libraries from an AT-rich genome with comparable coverage from 500 ng down to 10 pg input.

Evenness of Coverage for GC-rich Genome

  • A PCR-free library was prepared from B. pertussis genomic DNA (68% GC) using the Accel-NGS 2S DNA Library Kit (500 ng).
  • Left: Cumulative fraction of genome covered vs. depth of coverage, with theoretical perfect result (red) when entire genome is at normalized depth
  • Right: GC content of each read vs. theoretical in silico distribution and coverage

Conclusion. The Accel-NGS 2S DNA Library Kit produces libraries from a GC-rich genome with even coverage.

Evenness of Coverage for Extreme AT-rich Genome

  • A PCR-Free library was prepared from P. falciparum genomic DNA (19% GC) using the Accel-NGS 2S DNA Library Kit (500 ng)
  • Left: Cumulative fraction of genome covered vs. depth of coverage, with theoretical perfect result (red) when entire genome is at normalized depth
  • Right: GC content of each read vs. theoretical in silico distribution and coverage

Conclusion. The Accel-NGS DNA Library Kit produces libraries from an extremely AT-rich genome with even coverage.

Brochure

Click here to download brochure

Technical Resources

Click here to go to the “SWIFT BIOSCIENCES” Technical Resources