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Accel-NGS® 2S Hyb DNA Library Kit

Next-Generation Sequencing Prep for Hybridization Capture

Minimize Duplicates, Maximize Sequencing Data

The Accel-NGS 2S Hyb DNA Library Kit, designed for Illumina® platforms, produces 45% fewer duplicates at 1 ng to save you money on sequencing costs. A variety of indexing kits allow for compatibility with multiple hybridization capture technologies: Agilent SureSelect, NimbleGen SeqCap EZ, and IDT® xGen® Lockdown® Probes. This kit is the right choice for researchers working with applications pertaining to low input FFPE DNA or whole genome sequencing of liquid biopsy and cfDNA samples.

Features:

  • Sequential repair steps
  • Superior library preparation efficiency
  • Exceptional library complexity
  • Balanced coverage of AT-/GC-rich regions
  • No adapter titration or heat steps involved

Benefits:

  • Compatibility with limiting FFPE and cfDNA samples
  • More unique molecules available for capture
  • Meaningful data from low input samples
  • Suitable for a diverse set of panels
  • Readily automatable

2s-hyb-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.

Determine which Accel-NGS 2S Hyb Indexing Kit is right for your application.

The Accel-NGS 2S Hyb Kit does not contain a polymerase, but does contain amplification primers that can be used to amplify these libraries with a polymerase of your choice.

2s-hyb-decision-tree

† Please use the SureSelectXT and SureSelectXT2 Hybridization Capture Compatibility with the Accel-NGS 2S Hyb Library Kit Technical Note for details.
*We recommend using the Accel-NGS 2S Hyb to construct libraries. For amplification of these libraries, we advise using the polymerase validated with the hybridization capture reagents.

If you have any questions, please contact Tech Support.

Other Accel-NGS 2S Kits are available for users not performing hybridization capture. For users with at least 100 ng of consistently high quality DNA performing direct sequencing, use the Accel-NGS 2S PCR-Free DNA Library Kit. For users with less than 100 ng of DNA or low quality DNA performing direct sequencing, use the Accel-NGS 2S Plus DNA Library Kit.

Performance with Limiting Samples on the Illumina Platform

The data below demonstrate the performance of the Accel-NGS 2S Hyb DNA Library Kit with various hybridization capture panels with low input and FFPE samples.

Consistent Coverage of Low Input FFPE Samples

To compare the performance of the Accel-NGS 2S Hyb kit and the IDT xGen AML Cancer Panel with high quality DNA and damaged DNA from FFPE samples, multiple input quantites were evaluated: 100, 10 and 1 ng of high quality DNA (NA12878), and 10 and 1 ng of DNA from FFPE samples and a formalin compromised Horizon Discovery reference standard (HDx FC). Reads were normalized to 1.1M for comparision of coverage metrics.

Coverage of AML Cancer Panel with 100, 10 and 1 ng Inputs

Prod_2S Hyb_Table_AML Cancer Panel

Accel-NGS 2S Hyb kit performance with the IDT xGen AML Cancer Panel was evaluated with 100, 10, and 1 ng inputs of high quality Coriell NA12878 gDNA, and 10 and 1 ng inputs of FFPE samples and a formalin-compromised HDx reference standard.
Increased Coverage and Accurate SNP Variant Calls with Limiting Samples

Accel-NGS 2S Hyb kit and Kapa Library Preparation kit performance was compared with the NimbleGen SeqCap EZ MedExome Panel. High quality Coriell NA12878 gDNA at 100, 10 and 1 ng inputs were evaluated. Reads were normalized to 39M for comparison of coverage metrics. SNP concordance with the NIST GIAB truth list in high-confidence regions was >99% for all inputs and library preparation methods, with the exception of Kapa 1 ng, which was 98% (Zook et al Nature Biotechnology 2014). Kapa Library Preparation performance drops significantly at 1 ng, illustrated by duplication rates, mean coverage depth, percentage of the target area covered at least 20X, and the sensitivity/precision of SNP variant calls (as shown in the graphs below).

Superior Performance with the MedExome Panel

Prod_2S Hyb_Table_MedExome Panel

Libraries were made from 100 , 10, and 1 ng of HapMap DNA NA12878 (Coriell) with both the Swift Accel-NGS 2S Hyb kit and the Kapa Library Preparation kit, followed by the NimbleGen SeqCap EZ MedExome Panel. All libraries were normalized to 39M reads.

Exceptional Coverage and SNP Calls

2S Hyb_SNP Calls

Minimal Duplication Rates and Superior Coverage of Pan-Cancer Panel

The Accel-NGS 2S Hyb kit and the IDT xGen Pan-Cancer Panel were evaluated with samples from a variety of input quantities and qualities. High quality Coriell DNA (NA12878) performance can be compared to DNA samples from cfDNA, formalin-compromised standards, and normal kidney FFPE samples that have undergone 6 to 48 hours of formalin fixation. Formalin-compromised standards displayed a range of duplication rates and depths of coverage, suggesting that the usable amount of DNA within a sample can be greatly affected by sample quality.

High Quality Genomic DNA with the Pan-Cancer Panel

Prod_2S Hyb_Table_Genomic DNA with Pan-Cancer

Accel-NGS 2S Hyb libraries were constructed from 100, 10 and 1 ng of Coriell HapMap NA12878 DNA. Libraries were enriched with the IDT xGen Pan-Cancer Panel.
Cell-Free DNA with the Pan-Cancer Panel

Prod_2S Hyb_Table_cfDNA with Pan-Cancer

Accel-NGS 2S Hyb libraries were constructed from 15 and 5 ng of circulating, cell-free DNA. Libraries were enriched with the IDT xGen Pan-Cancer Panel.
Formalin-Compromised DNA Samples with the Pan-Cancer Panel

Prod_2S Hyb_Table_Formalin with Pan-Cancer

Accel-NGS 2S Hyb libraries were constructed from 5 and 1 ng of Horizon Discovery standards. HDx701 is not a fixed sample. HDx749 and HDx751 are formalin compromised versions of the same DNA present in HDx701. Libraries were enriched with the IDT xGen Pan-Cancer Panel.
Fixation Time Course with the Pan-Cancer Panel

Prod_2S Hyb_Table_Fixation with Pan-Cancer

Accel-NGS 2S Hyb libraries were constructed from 100, 10 and 1 ng of DNA extracted from normal kidney samples that had been frozen or subjected to 6, 24 or 48 hours of fixation. Libraries were enriched with the IDT xGen Pan-Cancer Panel.

To request sequencing data files (FASTQ, BAM, and VCF) for the Accel-NGS 2S Hyb DNA Library Kit, please email TechSupport@swiftbiosci.com.

Protocols and Tools

Application Note

Product Overview

Scientific Posters

Technical Notes

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
23024 Accel-NGS 2S Hyb DNA Library Kit (24) $645.00

$645.00 Add to cart

23096 Accel-NGS 2S Hyb DNA Library Kit (96) $2375.00

$2,375.00 Add to cart

For Agilent SureSelect Target Enrichment, choose:

Catalog No. Description Price QTY
26424  SureSelect Compatibility Module (24 rxns) $120.00

$120.00 Add to cart

26496  SureSelect Compatibility Module (96 rxns) $480.00

$480.00 Add to cart

27424  SureSelect MID Compatibility Module (24 rxns) $192.00

$192.00 Add to cart

27496  SureSelect MID Compatibility Module (96 rxns) $768.00

$768.00 Add to cart

For NimbleGen SeqCap EZ Target Enrichment, choose:

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

For IDT xGen Lockdown Probes, choose:

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

28096  2S Dual Indexing Kit (96 unique combinations) $480.00

$480.00 Add to cart

The below solutions are included with your Library Kit. If you require extra, please add now:

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