Accel-NGS® 2S Plus DNA Library Kit
Improve DNA Sequencing Results by Covering More of the Genome
The Accel-NGS 2S Plus DNA Library Kit, designed for Illumina platforms, utilizes a proprietary adapter attachment chemistry which minimizes bias and supports inputs as low as 10 pg. The Accel-NGS 2S Plus kit offers exceptional performance for a broad range of applications pertaining to whole genome sequencing of limiting samples such as FFPE and ChIP-Seq.
- Exceptional yields from DNA inputs of 10 pg to 250 ng
- Sequential repair steps
- High complexity libraries for deep sequencing
- No base composition bias from AT-/GC-rich genomes
- No adapter titration or heat steps involved
- “With bead” workflow
- Reduce concerns of proper quantification
- Excellent library preparation efficiency
- More complete genome coverage
- Better data quality, and evenness of coverage
- Readily automatable
- Reduce workflow costs and time
Using four incubations, this protocol repairs both 5’ and 3’ termini and sequentially attaches Illumina adapter sequences to the ends of fragmented dsDNA.
Bead-based clean-ups are used to remove oligonucleotides and small fragments, and to change enzymatic buffer composition between steps. Different bead-to-sample ratios are utilized for different input quantities and insert sizes.
For PCR-free applications, the resulting functional library is ready for quantification and sequencing on the Illumina platform. Alternatively, an optional PCR step may be used to increase yield of indexed libraries, which then may be quantified and sequenced.
Accel-NGS 2S Plus Workflow for Single Indexing Kits and Single Indexed Adapters (Ordering Information)
Accel-NGS 2S Plus Workflow for Single Indexing Kits with MIDs and Dual Indexed Adapters (Ordering Information)
Accel-NGS 2S Plus Workflow for Dual Indexing Kits (Cat. No. 28096 only)
Determine which Accel-NGS 2S Library Kit and Indexing Kit is the best choice for your application.
The Accel-NGS 2S Plus Kit contains a Swift Biosciences-validated polymerase for library amplification. The Accel-NGS 2S PCR-free Kit does not contain a polymerase, but does contain amplification primers, so that you may still amplify these libraries with a polymerase of your choice, if necessary.
Please see the below decision tree to determine the appropriate 2S Kit for your direct sequencing application.
†PCR free capability is determined by sample quality and input quantity. Accel-NGS 2S PCR-free libraries can be generated from as low as 100 ng of high quality genomic DNA (for < 100 ng inputs, see “Accel-NGS 2S DNA Library Kit for PCR-Free Libraries from 10 ng Input” Technical Note), or 10 ng of circulating, cell-free DNA.
*Multiplexing greater than 24 libraries requires the Accel-NGS 2S Dual Indexing Kit, which adds indexing sequences by PCR.
If you prefer to use your own polymerase and require multiplexing of more than 24 libraries, please contact Tech Support.
Sequence DNA More Efficiently
Accel-NGS 2S Plus 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 dynamic range of input levels. The kit’s broad input range means that pre-library quantification is optional, further streamlining the workflow. Finally, the Accel-NGS 2S Plus DNA Library Kit protocol is readily automatable for those with high throughput applications.
Performance with Microbial and Human gDNA on the Illumina Platform
The data below demonstrate the performance of the Accel-NGS 2S Plus DNA Library Kit with microbial and human genomic DNA.
Accel-NGS 2S Plus Produces Exceptional Yields from 10 pg
The Accel-NGS 2S Plus Kit chemistry is capable of processing a broad range of input quantities, including ultra-low input. Efficient library yields and minimized adapter dimer and duplicates for input quantities as low as 10 pg enables meaningful analysis of previously un-sequencable samples. Unlike some competitors, the Accel-NGS 2S Plus Kit does not require precise titration of adapters based on input amounts.
Accel-NGS 2S Plus library yields from 10 pg, 100 pg and 1 ng of E. coli DNA. Prepared library yields were determined by qPCR quantification.
Accel-NGS 2S Plus Compatibilty with Ultra Low Inputs
Limiting samples and inefficient extraction methods can result in ultra low quantities of sample DNA available for sequencing. This reduction in usable DNA for NGS library preparation can become a barrier to analysis, as low efficiency library preparations produce seqeuncing data containing regions of low or missing coverage. The highly efficient Accel-NGS 2S Plus Kit addresses the growing demand for a method that can convert a small amount of sample DNA into a high quality NGS library exhibiting even coverage.
Unlike many competitors’ products, which are not recommended or promoted for use with 10 pg of input DNA, the Accel-NGS 2S Plus Kit’s chemistry demonstrates excellent performance with ultra low input quantities. This makes the Accel-NGS 2S Plus Kit ideally suited for limited precious samples such as environmental samples containing low amounts of unculturable bacteria or small clinical samples, and it produces exceptional results even from low input quantities.
Accel-NGS 2S Plus libraries were constructed from 10 pg (left) or 10 ng (right) of DNA from an E. coli sample followed by 15 and 6 cycles of amplification, respectively. Sequencing was performed on an Illumina MiSeq®. Picard CollectGcBiasMetrics (picard.sourceforge.net) demonstrated evenness of coverage regardless of input amount and number of PCR cycles.
Comprehensive, Uniform Coverage from 1 ng of Human DNA
The Accel-NGS 2S Plus Kit also offers outstanding evenness of coverage for larger, complex genomes such as those from human. This holds true for both PCR-free and libraries requiring PCR amplification. The Accel-NGS 2S Plus Kit enables high quality libraries to be constructed from various inputs of human DNA without compromising the quality of the data.
Accel-NGS 2S Plus libraries were constructed from Coriell HapMap NA12878 DNA, 10 ng and 1 ng inputs. Sequencing was performed on an Illumina HiSeq® 2500. Data was analyzed using BWA (Li and Durbin, 2010) and Picard CollectGcBiasMetrics (picard.sourceforge.net). These Picard plots demonstrate that library constructed from 10 ng and 1 ng with an Accel-NGS 2S Plus Kit provide even coverage across a range of GC content.
Protocols and Tools
- Download Protocol
- Accel-NGS 2S Set A and B Indexing Kit Master Mix Volume Calculator
- Accel-NGS 2S Set S1-S4 Indexed Adapter Master Mix Volume Calculator
The Accel-NGS 2S kits are used for whole genome sequencing, de novo sequencing, whole exome sequencing, hybridization capture enrichment, metagenomics and ChIP-Seq applications.
The Accel-NGS 2S library kits are recommended for genomic DNA, FFPE, circulating, cell free DNA (cfDNA) and fresh frozen tissue samples.
Yes, we recommend using a qPCR-based assay to quantify starting material with amplicons that are sized to indicate the amplifiable content of the sample. There are several commercially-available qPCR-based input quantification kits available.
Yes, cfDNA recommendations are specified in the protocol, and include a modified Repair I Thermocycler program (to minimize chimera formation) and modified SPRI™ Cleanup Step bead and PEG NaCl volumes (for 2S PCR-Free and 2S Plus Kits only; to ensure capture of the relatively short cfDNA fragments).
The shelf life of the Accel-NGS 2S Plus, Accel-NGS 2S Hyb and Accel-NGS 2S PCR-free DNA Library kits is 6 months, when stored at -20C.
The recommended storage condition for the Accel-NGS 2S Plus and Accel-NGS 2S PCR-free DNA Library kits is -20C.
The sequences of the adapters in Accel-NGS 2S libraries are identical to Illumina TruSeq® LT adapters (Single Indexing) or Illumina TruSeq HT adapters (Dual Indexing), but are constructed in a proprietary manner. The adapters and indices are supplied directly from Swift Biosciences in a 2S Indexing Kit.
Oligonucleotide sequences © 2016 Illumina, Inc. All rights reserved.
No, the unique chemistry of the Accel-NGS 2S Kits maintains a low rate of adapter dimer formation without the need to titrate adapters.
Accel-NGS 2S adapters are constructed in a proprietary manner and must be purchased from Swift. The adapters and indices are supplied directly from Swift Biosciences.
Yes, for customers who prefer to use their own polymerase, we recommend the Accel-NGS 2S PCR-free DNA Library Kit. This kit contains amplification primers, but does not include a polymerase.
Enzymatic and sonication methods (Covaris) are supported for DNA fragmentation prior to library creation. For more information, refer to our Enzymatic Fragmentation with Accel-NGS 2S technical note.
The Accel-NGS 2S kits have been validated with 165 bp (cfDNA), 200 bp (gDNA), 350 bp (gDNA), and 450 bp (gDNA) fragments. If working with fragments of another size, please contact Tech Support for recommendations.
The minimal input for creating PCR-free libraries is 100ng of high quality DNA, 10ng of cfDNA, or 5ng when pooling samples. Please refer to our PCR-Free Libraries from 10 ng Input with the Accel-NGS 2S PCR-Free DNA Library Kit technical note for more details.
The input ranges for Accel-NGS 2S kits are 10pg – 250ng. Please consider genome complexity and sample quality when choosing input DNA quantity. Although libraries may be successfully prepared from ultra-low inputs, reduced representation of genome complexity may occur.
Accel-NGS 2S Kits construct high complexity libraries from dsDNA input through two dedicated repair steps and sequential ligation of adapters. Repair I dephosphorylates 5’ termini of input dsDNA to prevent chimera formation. Repair II performs 3’ end repair and polishing. Ligation I performs 3’ ligation of the P7 adapter, and Ligation II performs 5’ ligation of the P5 adapter. These separate, sequential ligation steps prevent formation of adapter dimers and enable independent optimization of each adapter attachment to each terminus. Following Ligation II, an Optional PCR Step can be performed to amplify the library, if necessary.
Repair I dephosphorylates 5’ termini of input dsDNA to prevent chimera formation. Chimeric library molecules can affect alignment metrics, as they are composed of fragments that will be read as a single library molecule, but will align to different genomic locations. Repair II performs 3’ end repair and polishing, which is critical to efficient ligation of adapters.
These separate, sequential ligation steps prevent formation of adapter dimers. As adapter dimers will take up space on the flow cell without providing meaningful sequencing data, minimizing adapter dimer formation during library preparation results in more efficient sample sequencing. Additionally, sequential ligation steps enable independent optimization of each adapter attachment to each terminus, which maximizes efficiency of library construction.
Yes, for customers that have not yet prepared the Ligation I master mix, we recommend pausing the library preparation following the Post-Repair II Step. We recommend re-suspending the SPRI beads in 10 µl of Low EDTA TE following the Post-Repair II SPRI Step, and storing the samples at 4°C. When you are ready to resume the library preparation, adjust the Ligation I master mix to include only 10 µl of Low EDTA TE rather than 20 µl. This will account for the 10 µl of Low EDTA TE that the beads have been stored in, so that the final volume of the Ligation I reaction remains the same. Proceed to run the samples with the Ligation I Thermocycler Program.
For customers that have already prepared the Ligation I master mix who desire a safe stopping point prior to the Post-Ligation II SPRI Step, please contact Tech Support for recommendations.
The Accel-NGS 2S Kits have been validated with SPRIselect beads. However, AMPure XP beads prove to exhibit equivalent performance and are an acceptable alternative.
Following the standard SPRI Cleanup steps that are specified in the protocol will result in left side size selection (removal of small DNA only). For customers with samples that contain large fragments (>600 bp), we recommend removal of these prior to library quantification as these large library molecules will contribute to the library concentration, but will not cluster well on the flow cell. For information on performing a right side size selection (removal of large DNA only) or double size selection (removal of both small and large DNA), please see the SPRIselect User Guide published by Beckman Coulter.
For high quality, fragmented gDNA, the Accel-NGS 2S Kits exhibit a conversion efficiency of 20-70%.* For circulating, cell-free DNA (cfDNA), higher conversion efficiencies are observed (up to 90%). This increase can be attributed to the relatively undamaged ends of cfDNA resulting from enzymatic cleavage in the blood and the narrow size distribution of cfDNA. For low quality DNA samples, such as those from FFPE, conversion rates can be lower due to unrepairable damage on the ends of DNA.
*Conversion efficiency calculations compare PCR-free yield (in nM) to the theoretical maximum yield (in nM). This calculation accounts for the added weight of NGS adapters, which can artificially inflate conversion efficiency.
Lower than expected yields can usually be attributed to inaccurate quantification of input DNA or inefficient recovery of DNA during the bead-based clean-up steps. While NanoDrop® or Qubit® may be acceptable for high quality DNA samples, quantification by a qPCR-based method is recommended to ensure accuracy of input DNA.
For damaged DNA samples, please be aware that small fragments (< 100 bp) will be excluded by the standard SPRI bead ratios indicated in the protocol.
There are two common causes for abnormal migration of Accel-NGS 2S library molecules.
The first cause applies to PCR-free libraries only. The secondary structure of sequencing adapters in PCR-free Accel-NGS 2S libraries results in abnormal migration and overestimation of library size. On the Agilent High Sensitivity Chip, it is normal and expected to observe 200 bp insert PCR-free libraries to migrate to a ~500 bp peak, and for 350 bp insert PCR-free libraries to migrate to a ~800 bp peak. Performing a few cycles of PCR on the library resolves the adapter secondary structure and results in library molecules that migrate true to size.
The second cause applies to libraries that have been over-amplified. Too many cycles of PCR can deplete primer concentration in the reaction, resulting in the formation of library molecule heteroduplex structures that will migrate abnormally. Denaturation of these heteroduplex structures – with a denaturing gel, for example – will result in library molecules that migrate true to size. Over-amplified libraries can still be sequenced, as they will be denatured just prior to loading on the flow cell, but customers should aim to perform the minimum number of PCR cycles necessary to avoid undesirable PCR duplicates.
PCR-free libraries must be quantified by qPCR to ensure accuracy. PCR-free libraries cannot be accurately quantified or assessed for library size on the Bioanalyzer.
96 different indices are currently available for Accel-NGS 2S products. Please consider your sample type, desired depth of sequencing, and sequencing instrument capabilities when determining your level of multiplexing.
Yes, Accel-NGS 2S kits are readily compatible with automation instruments. Scripts are in the process of being written for the Beckman Coulter Biomek® FXP and NXP, the PerkinElmer SciClone® and SciClone Janus®, the TECAN Fluent™, the Hamilton Microlab® STAR™, and the Eppendorf epMotion®.
Please contact Tech Support for more details.
The Accel-NGS 2S libraries are only compatible with Illumina platforms. However, Swift’s Accel-NGS DNA Library Kit for Ion Torrent will construct libraries compatible with Ion Torrent instrumentation.
First, select a product and quantity:
|21024||Accel-NGS 2S Plus DNA Library Kit (24 rxns)||$695.00|
|21096||Accel-NGS 2S Plus DNA Library Kit (96 rxns)||$2575.00|
Next, an Indexing Kit is required for complete functionality. Please select from the following:
|26148||2S Set A Indexing Kit (12 indices, 48 rxns)||$240.00|
|26248||2S Set B Indexing Kit (12 indices, 48 rxns)||$240.00|
|26396||2S Set A+B Indexing Kit (24 indices, 96 rxns)||$480.00|
|27148||2S Set A MID Indexing Kit (12 indices, 48 rxns)||$384.00|
|27248||2S Set B MID Indexing Kit (12 indices, 48 rxns)||$384.00|
|27396||2S Set A+B MID Indexing Kit (24 indices, 96 rxns)||$768.00|
|28096||2S Combinatorial Dual Indexing Kit (12 x 8)||$480.00|
The below solutions are included with your Library Kit. If you require extra, please add now:
|90196||PEG NaCl Solution (96 rxns)||$20.60|
|90296||Low EDTA TE (96 rxns)||$20.60|