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Accel-NGS® 2S PCR-Free DNA Library Kit

PCR-free NGS Prep with Low Input Capability for Illumina® Platforms

Eliminate Sequencing Bias from Polymerase with PCR-Free Preps

The Accel-NGS 2S PCR-Free DNA Library Kit, designed for Illumina platforms, enables preparation for high complexity NGS libraries from double-stranded DNA. PCR-free libraries can be made with unbiased coverage from cfDNA inputs as low as 10 ng and sheared DNA as low as 100 ng. The Accel-NGS 2S PCR-Free Kit offers greater than 90% conversion efficiency for applications pertaining to whole genome sequencing of relatively less damaged samples such as liquid biopsy and cfDNA. This technology delivers the following features and benefits:

Features:

  • PCR-free libraries from 10 ng cfDNA and 100 ng sheared DNA inputs
  • Sequential repair steps
  • Optional library amplification with self-selected polymerase
  • Superior library complexity
  • No adapter titration or heat steps involved

Benefits:

  • No base composition bias from AT-/GC-rich genomes
  • Excellent library preparation efficiency
  • Easily adaptable with other polymerase products
  • Generates more unique library molecules
  • Readily automatable

2s-pcrfree-workflow-100416

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. Bead-based SPRI clean-ups are used to remove oligonucleotides and small fragments, and to change enzymatic buffer composition between steps. This Protocol offers four incubation steps that repair both 5’ and 3’ termini and sequentially attach Illumina adapter sequences to the ends of fragmented dsDNA. For PCR-free applications, the resulting functional library is completed prior to the amplification step for library 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.

Determine which Accel-NGS 2S Library Kit and Indexing Kit is the best choice for your application.

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.

2s-decision-tree

†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.

NGS Library Yields Without Bias

The Accel-NGS 2S PCR-Free Kit offers the same outstanding sequence coverage as the Accel-NGS 2S Plus DNA Library Kit, allowing significant cost and time savings for labs wishing to perform PCR-free preps or enabling a different library enrichment or amplification strategy.

Performance with Human gDNA on the Illumina Platform

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

Save on sequencing costs by increasing library complexity

Increased library complexity from the Accel-NGS 2S PCR-Free DNA Library Kit improves sample coverage and reveals even difficult to read sequences in your NGS samples. Swift Biosciences utilizes a proprietary adapter ligation chemistry that provides more unique molecules per sample for sequencing. This means you increase useful sequence yield, from more samples, in a single instrument run. Download our latest application note to learn how increased library complexity generated from the Accel-NGS 2S PCR-Free and Accel-NGS 2S Plus DNA library kits help reduce overall sequencing costs.

Generate Higher Complexity Libraries than the Leading Competitive Kit

Swift Biosciences technology yields more unique library molecules than the competitive kit which allows for greater coverage of the genome in each sequencing run.

Library Complexity

Libraries were made PCR-free from HapMap DNA NA12878 (Coriell) and sequenced on the HiSeq. Competitor I data is from public access files. Estimated library size was calculated by Picard MarkDuplicates (picard.sourceforge.net).
Cover Extreme Base Composition Regions Better

With low bias of AT- and GC-rich regions, Swift Biosciences delivers increased coverage of the 1,000 Bad Promoters in human genome samples.

Bad Promoters

Libraries were made PCR-free from HapMap DNA NA12878 (Coriell) and sequenced on the HiSeq. Competitor I data is from public access files.
Expect Even Coverage with Human DNA Sequencing

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

Depth of Coverage_Human DNA

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 PCR-Free DNA Library Kit with microbial genomes of varying base composition that were prepared and sequenced on the Illumina MiSeq by a third party.

Evenness of Coverage for GC-rich Genome

Eveness of Coverage_GC-rich

  • A PCR-free library was prepared from B. pertussis genomic DNA (68% GC) using the Accel-NGS 2S PCR-free 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 PCR-free Kit produces libraries from a GC-rich genome with even coverage.

Evenness of Coverage for Extreme AT-rich Genome

Eveness of Coverage_AT-rich

  • A PCR-free library was prepared from P. falciparum genomic DNA (19% GC) using the Accel-NGS 2S PCR-free 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 PCR-free DNA Library Kit produces libraries from an extremely AT-rich genome with even coverage.

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.

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 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.

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.

Place Order

First, select a product and quantity:

Catalog No. Description Price QTY
20024 Accel-NGS 2S PCR-Free Library Kit (24 rxns) $645.00

$645.00 Add to cart

20096 Accel-NGS 2S PCR-Free Library Kit (96 rxns) $2375.00

$2,375.00 Add to cart

Next, an Indexing Kit is required for complete functionality. Please select from the following:

Catalog No. Description Price QTY
26148  2S Set A Indexing Kit (12 indices, 48 rxns) $240.00

$240.00 Add to cart

26248  2S Set B Indexing Kit (12 indices, 48 rxns) $240.00

$240.00 Add to cart

26396  2S Set A+B Indexing Kit (24 indices, 96 rxns) $480.00

$480.00 Add to cart

27148  2S Set A MID Indexing Kit (12 indices, 48 rxns) $384.00

$384.00 Add to cart

27248  2S Set B MID Indexing Kit (12 indices, 48 rxns) $384.00

$384.00 Add to cart

27396  2S Set A+B MID Indexing Kit (24 indices, 96 rxns) $768.00

$768.00 Add to cart

And, add optional components:

Catalog No. Description Price QTY
90196 PEG NaCl Solution (96 rxns) $20.00

$20.00 Add to cart

90296 Low EDTA TE (96 rxns) $20.00

$20.00 Add to cart